Determining a nucleic acid sequence imbalance using multiple markers

ABSTRACT

Methods, systems, and apparatus are provided for determining whether a nucleic acid sequence imbalance exists within a biological sample. One or more cutoff values for determining an imbalance of, for example, the ratio of the two sequences (or sets of sequences) are chosen. The cutoff value may be determined based at least in part on the percentage of fetal DNA in a sample, such as maternal plasma, containing a background of maternal nucleic acid sequences. The percentage of fetal DNA can be calculated from the same or different data used to determine the cutoff value, and can use a locus where the mother is homozygous and the fetus is heterozygous. The cutoff value may be determined using many different types of methods, such as sequential probability ratio testing (SPRT).

CLAIM OF PRIORITY

The present application is a divisional of U.S. application Ser. No.14/030,904 entitled “Determining Percentage Of Fetal DNA In MaternalSample” filed Sep. 18, 2013, which is a divisional of U.S. applicationSer. No. 12/178,116 entitled “Determining A Nucleic Acid SequenceImbalance” filed Jul. 23, 2008, now U.S. Pat. No. 8,706,422, whichclaims priority from and is a non-provisional application of U.S.Provisional Application No. 60/951,438, entitled “Determining A NucleicAcid Sequence Imbalance” filed Jul. 23, 2007, the entire contents ofwhich are herein incorporated by reference for all purposes.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is also related to U.S. application Ser. No.12/178,181 entitled “Diagnosing Fetal Chromosomal Aneuploidy UsingGenomic Sequencing,” filed Jul. 23, 2008, the entire contents of whichare herein incorporated by reference for all purposes.

REFERENCE TO A “SEQUENCE LISTING” SUBMITTED AS ASCII TEXT FILES VIAEFS-WEB

The Sequence Listing written in file080015-005213US-1120168_SequenceListing.txt created on Jan. 11, 2019,20,992 bytes, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§ 1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

FIELD

This invention generally relates to the diagnostic testing of genotypesand diseases by determining an imbalance between two different nucleicacid sequences, and more particularly to the identification of Downsyndrome, other chromosomal aneuploidies, mutations and genotypes in afetus via testing a sample of maternal blood. The invention also relatesto the detection of cancer, the monitoring of transplantation, and themonitoring of infectious diseases.

BACKGROUND

Genetic diseases, cancers, and other conditions often result from orproduce an imbalance in two corresponding chromosomes or alleles orother nucleic acid sequences. That is an amount of one sequence relativeto another sequence is larger or smaller than normal. Usually, thenormal ratio is an even 50/50 ratio. Down Syndrome (trisomy 21) is sucha disease having an imbalance of an extra chromosome 21.

Conventional prenatal diagnostic methods of trisomy 21 involve thesampling of fetal materials by invasive procedures such as amniocentesisor chorionic villus sampling, which pose a finite risk of fetal loss.Non-invasive procedures, such as screening by ultrasonography andbiochemical markers, have been used to risk-stratify pregnant womenprior to definitive invasive diagnostic procedures. However, thesescreening methods typically measure epiphenomena that are associatedwith trisomy 21 instead of the core chromosomal abnormality, and thushave suboptimal diagnostic accuracy and other disadvantages, such asbeing highly influenced by gestational age.

The discovery of circulating cell-free fetal DNA in maternal plasma in1997 offered new possibilities for noninvasive prenatal diagnosis (Lo, YM D and Chiu, R W K 2007 Nat Rev Genet 8, 71-77). While this method hasbeen readily applied to the prenatal diagnosis of sex-linked (Costa, J Met al. 2002 N Engl J Med 346, 1502) and certain single gene disorders(Lo, Y M D et al. 1998 N Engl J Med 339, 1734-1738), its application tothe prenatal detection of fetal chromosomal aneuploidies has representeda considerable challenge (Lo, Y M D and Chiu, R W K 2007, supra). First,fetal nucleic acids co-exist in maternal plasma with a high backgroundof nucleic acids of maternal origin that can often interfere with theanalysis (Lo, Y M D et al. 1998 Am J Hum Genet 62, 768-775). Second,fetal nucleic acids circulate in maternal plasma predominantly in acell-free form, making it difficult to derive dosage information ofgenes or chromosomes within the fetal genome.

Significant developments overcoming these challenges have recently beenmade (Benachi, A & Costa, J M 2007 Lancet 369, 440-442). One approachdetects fetal-specific nucleic acids in the maternal plasma, thusovercoming the problem of maternal background interference (Lo, Y M Dand Chiu, R W K 2007, supra). Dosage of chromosome 21 was inferred fromthe ratios of polymorphic alleles in the placenta-derived DNA/RNAmolecules. However, this method is less accurate when samples containlower amount of the targeted gene and can only be applied to fetuses whoare heterozygous for the targeted polymorphisms, which is only a subsetof the population if one polymorphism is used.

Dhallan et al (Dhallan, R, et al. 2007, supra Dhallan, R, et al. 2007Lancet 369, 474-481) described an alternative strategy of enriching theproportion of circulating fetal DNA by adding formaldehyde to maternalplasma. The proportion of chromosome 21 sequences contributed by thefetus in maternal plasma was determined by assessing the ratio ofpaternally-inherited fetal-specific alleles to non-fetal-specificalleles for single nucleotide polymorphisms (SNPs) on chromosome 21. SNPratios were similarly computed for a reference chromosome. An imbalanceof fetal chromosome 21 was then inferred by detecting a statisticallysignificant difference between the SNP ratios for chromosome 21 andthose of the reference chromosome, where significant is defined using afixed p-value of ≤0.05. To ensure high population coverage, more than500 SNPs were targeted per chromosome. However, there have beencontroversies regarding the effectiveness of formaldehyde to enrich to ahigh proportion (Chung, G T Y, et al. 2005 Clin Chem 51, 655-658), andthus the reproducibility of the method needs to be further evaluated.Also, as each fetus and mother would be informative for a differentnumber of SNPs for each chromosome, the power of the statistical testfor SNP ratio comparison would be variable from case to case (Lo, Y M D& Chiu, R W K. 2007 Lancet 369, 1997). Furthermore, since theseapproaches depend on the detection of genetic polymorphisms, they arelimited to fetuses heterozygous for these polymorphisms.

Using polymerase chain reaction (PCR) and DNA quantification of achromosome 21 locus and a reference locus in amniocyte cultures obtainedfrom trisomy 21 and euploid fetuses, Zimmermann et al (2002 Clin Chem48, 362-363) were able to distinguish the two groups of fetuses based onthe 1.5-fold increase in chromosome 21 DNA sequences in the former.Since a 2-fold difference in DNA template concentration constitutes adifference of only one threshold cycle (Ct), the discrimination of a1.5-fold difference has been the limit of conventional real-time PCR. Toachieve finer degrees of quantitative discrimination, alternativestrategies are needed. Accordingly, some embodiments of the presentinvention use digital PCR (Vogelstein, B et al. 1999 Proc Natl Acad SciUSA 96, 9236-9241) for this purpose.

Digital PCR has been developed for the detection of allelic ratioskewing in nucleic acid samples (Chang, H W et al. 2002 J Natl CancerInst 94, 1697-1703). Clinically, it has been shown to be useful for thedetection of loss of heterozygosity (LOH) in tumor DNA samples (Zhou, W.et al. 2002 Lancet 359, 219-225). For the analysis of digital PCRresults, sequential probability ratio testing (SPRT) has been adopted byprevious studies to classify the experimental results as beingsuggestive of the presence of LOH in a sample or not (El Karoui at al.2006 Stat Med 25, 3124-3133). In methods used in the previous studies,the cutoff value to determine LOH used a fixed reference ratio of thetwo alleles in the DNA of 2/3. As the amount, proportion, andconcentration of fetal nucleic acids in maternal plasma are variable,these methods are not suitable for detecting trisomy 21 using fetalnucleic acids in a background of maternal nucleic acids in maternalplasma.

It is desirable to have a noninvasive test for fetal trisomy 21 (andother imbalances) detection based on circulating fetal nucleic acidanalysis, especially one that is independent of the use of geneticpolymorphisms and/or of fetal-specific markers. It is also desirable tohave accurate determination of cutoff values and counting of sequences,which can reduce the number of wells of data and/or the amount ofmaternal plasma nucleic acid molecules necessary for accuracy, thusproviding increased efficiency and cost-effectiveness. It is alsodesirable that noninvasive tests have high sensitivity and specificityto minimize false diagnoses.

Another application for fetal DNA detection in maternal plasma is forthe prenatal diagnosis of single gene disorders such asbeta-thalassemia. However, as fetal DNA only constitutes a minorfraction of DNA in maternal plasma, it is thought that this approach canonly detect a mutation that a fetus has inherited from its father, butwhich is absent from the mother. Examples of this include the 4 bpdeletion in codon 41/42 of the beta-globin gene causing beta-thalassemia(Chiu R W K et al. 2002 Lancet, 360, 998-1000) and the Q890X mutation ofthe cystic fibrosis transmembrance conductance regulator gene causingcystic fibrosis (Gonzalez-Gonzalez et al 2002 Prenat Diagn, 22, 946-8).However, as both beta-thalassemia and cystic fibrosis are autosomalrecessive conditions, in which the fetus would need to inherit amutation from each parent before the disease would manifest itself, thedetection of merely the paternally-inherited mutation would onlyincrease the risk of having the fetus having the disease from 25% to50%. Diagnostically this is not ideal. Thus, the main diagnosticapplication of the existing approach would be for the scenario when nopaternally-inherited fetal mutation can be detected in maternal plasma,when the fetus can then be excluded from having the homozygous diseasestate. However, diagnostically, this approach has the disadvantage thatthe conclusion is made based on the negative detection of the paternalmutation. Thus, an approach which would allow the complete fetalgenotype (be it homozygous normal, homozygous mutant, or heterozygous)to be determined from maternal plasma, without the above limitation,would be very desirable.

BRIEF SUMMARY

Embodiments of this invention provides methods, systems, and apparatusfor determining whether a nucleic acid sequence imbalance (e.g., allelicimbalance, mutational imbalance, or chromosome imbalance) exists withina biological sample. One or more cutoff values for determining animbalance of, for example, a ratio of amounts of the two sequences (orsets of sequences) are chosen.

In one embodiment, the cutoff value is determined based at least in parton the percentage of fetal (clinically relevant nucleic acid) sequencesin a biological sample, such as maternal plasma or serum or urine, whichcontains a background of maternal nucleic acid sequences. In anotherembodiment, the cutoff value is determined based on an averageconcentration of a sequence in a plurality of reactions. In one aspect,the cutoff value is determined from a proportion of informative wellsthat are estimated to contain a particular nucleic acid sequence, wherethe proportion is determined based on the above-mentioned percentageand/or average concentration.

The cutoff value may be determined using many different types ofmethods, such as SPRT, false discovery, confidence interval, receiveroperating characteristic (ROC). This strategy further minimized theamount of testing required before confident classification could bemade. This is of particular relevance to plasma nucleic acid analysiswhere the template amount is often limiting.

According to one exemplary embodiment, a method is provided fordetermining whether a nucleic acid sequence imbalance exists within abiological sample, the method comprising: receiving data from aplurality of reactions, wherein the data includes: (1) a first set ofquantitative data indicating a first amount of a clinically relevantnucleic acid sequence; and (2) a second set of quantitative dataindicating a second amount of a background nucleic acid sequencedifferent from the clinically relevant nucleic acid sequence;determining a parameter from the two data sets; deriving a first cutoffvalue from an average concentration of a reference nucleic acid sequencein each of the plurality of reactions, wherein the reference nucleicacid sequence is either the clinically relevant nucleic acid sequence orthe background nucleic acid sequence; comparing the parameter to thefirst cutoff value; and based on the comparison, determining aclassification of whether a nucleic acid sequence imbalance exists.

According to another exemplary embodiment, a method is provided fordetermining whether a nucleic acid sequence imbalance exists within abiological sample, the method comprising: receiving data from aplurality of reactions, wherein the data includes: (1) a first set ofquantitative data indicating a first amount of a clinically relevantnucleic acid sequence; and (2) a second set of quantitative dataindicating a second amount of a background nucleic acid sequencedifferent from the clinically relevant nucleic acid sequence, whereinthe clinically relevant nucleic acid sequence and the background nucleicacid sequence come from a first type of cells and from one or moresecond types of cells; determining a parameter from the two data sets;deriving a first cutoff value from a first percentage resulting from ameasurement of an amount of a nucleic acid sequence from the first typeof cells in the biological sample; comparing the parameter to the cutoffvalue; and based on the comparison, determining a classification ofwhether a nucleic acid sequence imbalance exists.

Other embodiments of the invention are directed to systems and computerreadable media associated with methods described herein.

A better understanding of the nature and advantages of the presentinvention may be gained with reference to the following detaileddescription and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart illustrating a digital PCR experiment.

FIG. 2A illustrates a digital RNA-SNP and RCD method according to anembodiment of the present invention.

FIG. 2B shows a table of examples of frequently detectable chromosomalaberrations in cancers.

FIG. 3 illustrates a graph having SPRT curves used to determine Downsyndrome according to an embodiment of the present invention.

FIG. 4 shows a method of determining a disease state using a percentageof fetal cells according to an embodiment of the present invention.

FIG. 5 shows a method of determining a disease state using an averageconcentration according to an embodiment of the present invention.

FIG. 6 shows a table that tabulates the expected digital RNA-SNP allelicratio and P_(r) of trisomy 21 samples for a range of templateconcentrations expressed as the average reference template concentrationper well (m_(r)) according to an embodiment of the present invention.

FIG. 7 shows a table that tabulates the expected P_(r) for thefractional fetal DNA concentrations of 10%, 25%, 50% and 100% in trisomy21 samples at a range of template concentrations expressed as theaverage reference template concentration per well (m_(r)) according toan embodiment of the present invention.

FIG. 8 shows a plot illustrating the degree of differences in the SPRTcurves for m_(r) values of 0.1, 0.5 and 1.0 for digital RNA-SNP analysisaccording to an embodiment of the present invention.

FIG. 9A shows a table of a comparison of the effectiveness of the newand old SPRT algorithms for classifying euploid and trisomy 21 cases in96-well digital RNA-SNP analyses according to an embodiment of thepresent invention.

FIG. 9B shows a table of a comparison of the effectiveness of the newand old SPRT algorithms for classifying euploid and trisomy 21 cases in384-well digital RNA-SNP analyses according to an embodiment of thepresent invention.

FIG. 10 is a table showing the percentages of fetuses correctly andincorrectly classified as euploid or aneuploid and those notclassifiable for the given informative counts according to an embodimentof the present invention.

FIG. 11 is a table 1100 showing computer simulations for digital RCDanalysis for a pure (100%) fetal DNA sample according to an embodimentof the present invention.

FIG. 12 is a table 1200 showing results of computer simulation ofaccuracies of digital RCD analysis at m_(r)=0.5 for the classificationof samples from euploid or trisomy 21 fetuses with different fractionalconcentrations of fetal DNA according to an embodiment of the presentinvention.

FIG. 13A shows a table 1300 of digital RNA-SNP analysis in placentaltissues of euploid and trisomy 21 pregnancies according to an embodimentof the present invention.

FIG. 13B shows a table 1350 of digital RNA-SNP analysis of maternalplasma from euploid and trisomy 21 pregnancies according to anembodiment of the present invention.

FIGS. 14A-14C show plots illustrating a cutoff curve resulting from anRCD analysis according to an embodiment of the present invention.

FIG. 15A shows a table of digital RNA-SNP analysis in placental tissuesof euploid and trisomy 21 pregnancies according to an embodiment of thepresent invention.

FIG. 15B shows a table of digital RNA-SNP data of the 12 reaction panelsfrom one maternal plasma sample according to an embodiment of thepresent invention.

FIG. 15C shows a table of digital RNA-SNP analysis of maternal plasmafrom euploid and trisomy 21 pregnancies according to an embodiment ofthe present invention.

FIG. 16A shows a table for a digital RNA-SNP analysis of euploid andtrisomy 18 placentas according to an embodiment of the presentinvention.

FIG. 16B shows an SPRT interpretation of digital RNA-SNP data foreuploid and trisomy 18 placentas according to an embodiment of thepresent invention.

FIG. 17 shows a table of a digital RCD analysis of 50%placental/maternal blood cell DNA mixtures of euploid and trisomy 21pregnancies according to an embodiment of the present invention.

FIG. 18 shows a SPRT curve illustrating the decision boundaries forcorrect classification according to an embodiment of the presentinvention.

FIG. 19 shows a table of digital RCD analysis of amniotic fluid samplesfrom euploid and trisomy 21 pregnancies according to an embodiment ofthe present invention.

FIG. 20 shows a table of digital RCD analysis of placental DNA samplesfrom euploid and trisomy 18 pregnancies (E=euploid; T18=trisomy 18)according to an embodiment of the present invention.

FIGS. 21A and 22B show a table of a multiplex digital RCD analysis of50% placental/maternal blood cell DNA mixtures of euploid and trisomy 21pregnancies (E=euploid; T21=trisomy 21; U=unclassified) according to anembodiment of the present invention.

FIGS. 22A and 22B show a table of a multiplex digital RCD analysis of50% euploid or trisomy 21 placental genomic DNA/50% maternal buffy coatDNA mix according to an embodiment of the present invention. Unclassdenotes unclassifiable and T21 denotes trisomy 21.

FIG. 23 shows a scenario where both the male and female partners carrythe same mutation.

FIG. 24A shows a table of a digital RMD analysis of female/male andmale/male DNA mixtures according to an embodiment of the presentinvention.

FIG. 24B shows a table of a digital RMD analysis of mixtures with 25%female and 75% male DNA according to an embodiment of the presentinvention.

FIG. 25 shows a table of a digital RMD analysis of 15%-50% DNA mixturesmimicking maternal plasma samples for HbE mutation according to anembodiment of the present invention.

FIG. 26A shows a table of a digital RMD analysis of 5%-50% DNA mixturesmimicking maternal plasma samples for CD41/42 mutation according to anembodiment of the present invention.

FIG. 26B shows a table of a digital RMD analysis of 20% DNA mixturesmimicking maternal plasma samples for CD41/42 mutation according to anembodiment of the present invention.

FIG. 27 shows a block diagram of an exemplary computer apparatus usablewith system and methods according to embodiments of the presentinvention.

DEFINITIONS

The term “biological sample” as used herein refers to any sample that istaken from a subject (e.g., a human, such as a pregnant woman) andcontains one or more nucleic acid molecule(s) of interest.

The term “nucleic acid” or “polynucleotide” refers to a deoxyribonucleicacid (DNA) or ribonucleic acid (RNA) and a polymer thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogs of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, small noncoding RNA, micro RNA(miRNA), Piwi-interacting RNA, and short hairpin RNA (shRNA) encoded bya gene or locus.

The term “gene” means the segment of DNA involved in producing apolypeptide chain. It may include regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “reaction” as used herein refers to any process involving achemical, enzymatic, or physical action that is indicative of thepresence or absence of a particular polynucleotide sequence of interest.An example of a “reaction” is an amplification reaction such as apolymerase chain reaction (PCR). Another example of a “reaction” is asequencing reaction, either by synthesis or by ligation. An “informativereaction” is one that indicates the presence of one or more particularpolynucleotide sequence of interest, and in one case where only onesequence of interest is present. The term “well” as used herein refersto a reaction at a predetermined location within a confined structure,e.g., a well-shaped vial, cell, or chamber in a PCR array.

The term “clinically relevant nucleic acid sequence” as used herein canrefer to a polynucleotide sequence corresponding to a segment of alarger genomic sequence whose potential imbalance is being tested or tothe larger genomic sequence itself. One example is the sequence ofchromosome 21. Other examples include chromosome 18, 13, X and Y. Yetother examples include mutated genetic sequences or geneticpolymorphisms or copy number variations that a fetus may inherit fromone or both of its parents. Yet other examples include sequences whichare mutated, deleted, or amplified in a malignant tumor, e.g. sequencesin which loss of heterozygosity or gene duplication occur. In someembodiments, multiple clinically relevant nucleic acid sequences, orequivalently multiple makers of the clinically relevant nucleic acidsequence, can be used to provide data for detecting the imbalance. Forinstance, data from five non-consecutive sequences on chromosome 21 canbe used in an additive fashion for the determination of a possiblechromosomal 21 imbalance, effectively reducing the need of sample volumeto 1/5.

The term “background nucleic acid sequence” as used herein refers to anucleic acid sequence whose normal ratio to the clinically relevantnucleic acid sequence is known, for instance a 1-to-1 ratio. As oneexample, the background nucleic acid sequence and the clinicallyrelevant nucleic acid sequence are two alleles from the same chromosomethat are distinct due to heterozygosity. In another example, thebackground nucleic acid sequence is one allele that is heterozygous toanother allele that is the clinically relevant nucleic acid sequence.Moreover, some of each of the background nucleic acid sequence and theclinically relevant nucleic acid sequence may come from differentindividuals.

The term “reference nucleic acid sequence” as used herein refers to anucleic acid sequence whose average concentration per reaction is knownor equivalently has been measured.

The term “overrepresented nucleic acid sequence” as used herein refersto the nucleic acid sequence among two sequences of interest (e.g., aclinically relevant sequence and a background sequence) that is in moreabundance than the other sequence in a biological sample.

The term “based on” as used herein means “based at least in part on” andrefers to one value (or result) being used in the determination ofanother value, such as occurs in the relationship of an input of amethod and the output of that method. The term “derive” as used hereinalso refers to the relationship of an input of a method and the outputof that method, such as occurs when the derivation is the calculation ofa formula.

The term “quantitative data” as used herein means data that are obtainedfrom one or more reactions and that provide one or more numericalvalues. For example, the number of wells that show a fluorescent markerfor a particular sequence would be quantitative data.

The term “parameter” as used herein means a numerical value thatcharacterizes a quantitative data set and/or a numerical relationshipbetween quantitative data sets. For example, a ratio (or function of aratio) between a first amount of a first nucleic acid sequence and asecond amount of a second nucleic acid sequence is a parameter.

The term “cutoff value” as used herein means a numerical value whosevalue is used to arbitrate between two or more states (e.g. diseased andnon-diseased) of classification for a biological sample. For example, ifa parameter is greater than the cutoff value, a first classification ofthe quantitative data is made (e.g. diseased state); or if the parameteris less than the cutoff value, a different classification of thequantitative data is made (e.g. non-diseased state).

The term “imbalance” as used herein means any significant deviation asdefined by at least one cutoff value in a quantity of the clinicallyrelevant nucleic acid sequence from a reference quantity. For example,the reference quantity could be a ratio of 3/5, and thus an imbalancewould occur if the measured ratio is 1:1.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods, systems, and apparatus for determiningwhether an increase or decrease compared to a reference (e.g.non-diseased) quantity of a clinically-relevant nucleic acid sequence inrelation to other non-clinically-relevant sequences (e.g., a chromosomalor allelic imbalance) exists within a biological sample. One or morecutoff values are chosen for determining whether a change compared tothe reference quantity exists (i.e. an imbalance), for example, withregards to the ratio of amounts of two sequences (or sets of sequences).The change detected in the reference quantity may be any deviation(upwards or downwards) in the relation of the clinically-relevantnucleic acid sequence to the other non-clinically-relevant sequences.Thus, the reference state may be any ratio or other quantity (e.g. otherthan a 1-1 correspondence), and a measured state signifying a change maybe any ratio or other quantity that differs from the reference quantityas determined by the one or more cutoff values.

The clinically relevant nucleic acid sequence and the background nucleicacid sequence may come from a first type of cells and from one or moresecond types of cells. For example, fetal nucleic acid sequencesoriginating from fetal/placental cells are present in a biologicalsample, such as maternal plasma, which contains a background of maternalnucleic acid sequences originating from maternal cells. Thus, in oneembodiment, the cutoff value is determined based at least in part on apercentage of the first type of cells in a biological sample. Note thepercentage of fetal sequences in a sample may be determined by anyfetal-derived loci and not limited to measuring the clinically-relevantnucleic acid sequences. In another embodiment, the cutoff value isdetermined at least in part on the percentage of tumor sequences in abiological sample, such as plasma, serum, saliva or urine, whichcontains a background of nucleic acid sequences derived from thenon-malignant cells within the body.

In yet another embodiment, the cutoff value is determined based on anaverage concentration of a sequence in a plurality of reactions. In oneaspect, the cutoff value is determined from a proportion of informativewells that are estimated to contain a particular nucleic acid sequence,where the proportion is determined based on the above-mentionedpercentage and/or average concentration. The cutoff value may bedetermined using many different types of methods, such as SPRT, falsediscovery, confidence interval, receiver operating characteristic (ROC).This strategy further minimizes the amount of testing required beforeconfident classification can be made. This is of particular relevance toplasma nucleic acid analysis where the template amount is oftenlimiting. Although presented with respect to digital PCR, other methodsmay be used.

Digital PCR involves multiple PCR analyses on extremely dilute nucleicacids such that most positive amplifications reflect the signal from asingle template molecule. Digital PCR thereby permits the counting ofindividual template molecules. The proportion of positive amplificationsamong the total number of PCRs analyzed allows an estimation of thetemplate concentration in the original or non-diluted sample. Thistechnique has been proposed to allow the detection of a variety ofgenetic phenomena (Vogelstein, B et al. 1999, supra) and has previouslybeen used for the detection of loss of heterozygosity in tumor samples(Zhou, W. et al. 2002, supra) and in the plasma of cancer patients(Chang, H W et al. 2002, supra). Since template molecule quantificationby digital PCR does not rely on dose-response relationships betweenreporter dyes and nucleic acid concentrations, its analytical precisionshould theoretically be superior to that of real-time PCR. Hence,digital PCR could potentially allow the discrimination of finer degreesof quantitative differences between target and reference loci.

To test this, we first assessed if digital PCR could determine theallelic ratio of PLAC4 mRNA (Lo, Y M D, et al. 2007 Nat Med 13,218-223), a placental transcript from chromosome 21, in maternal plasmaand thereby distinguish trisomy 21 and euploid fetuses. This approach isreferred as the digital RNA-SNP method. We then evaluated whether theincreased precision of digital PCR would allow the detection of fetalchromosomal aneuploidies without depending on genetic polymorphisms. Wecall this digital relative chromosome dosage (RCD) analysis. The formerapproach is polymorphism-dependent but requires less precision inquantitative discrimination while the latter approach ispolymorphism-independent but requires a higher precision forquantitative discrimination.

I. Digital RNA-SNP

A. Overview

Digital PCR is capable of detecting the presence of allelic ratioskewing of two alleles in a DNA sample. For example, it has been used todetect loss of heterozygosity (LOH) in a tumor DNA sample. Assuming thatthere are two alleles in the DNA sample, namely A and G, and the Aallele would be lost in the cells with LOH. When LOH is present in 50%of cells in the tumor sample, the allelic ratio of G:A in the DNA samplewould be 2:1. However, if LOH is not present in the tumor sample, theallelic ratio of G:A would be 1:1.

FIG. 1 is a flowchart 100 illustrating a digital PCR experiment. In step110, the DNA sample is diluted and then distributed to separate wells.Note that the inventors have determined that some plasma nucleic acidspecies are already quite diluted in the original sample. Accordingly,there is no need for dilution for some templates, if they are alreadypresent at the necessary concentrations. In the previous studies (e.g.Zhou et al 2002, supra), a DNA sample is diluted to an extent such thatthe average concentration of a specific “template DNA” is approximately0.5 molecule of one of the two templates per well. Note that the term“template DNA” appears to refer to either the A or the G alleles, andthat there is no rationale provided for this specific concentration.

In step 120, in each well, a PCR process is carried out to detect the Aand/or the G allele simultaneously. In step 130, the markers in eachwell are identified (e.g. via fluorescence), e.g. A, G, A and G, orneither. In the absence of LOH, the abundance of the A and the G allelesin the DNA sample would be the same (one copy each per cell). Therefore,the probabilities of a well being positive for the A allele and for theG allele would be the same. This would be reflected by the similarnumbers of wells being positive for the A or the G alleles. However,when LOH is present in 50% or greater of cells in a tumor sample, theallelic ratio of the G and the A alleles would be at least 2:1. Previousmethods simply assumed that the sample was at least 50% cancerous. Thus,the probability of a well being positive for the G allele would behigher than that for the A allele. As a result, the number of wellsbeing positive for the G allele would be higher than that for the Aallele.

In step 140, to classify the digital PCR results, the number of wellsbeing positive for each allele but not the other would be counted. Inthe above example, the number of wells being positive for the A allelebut negative for the G allele, and the number of wells positive for theG allele but negative for the A allele are counted. In one embodiment,the allele showing less positive wells is regarded as the referenceallele.

In step 150, the total number of informative wells is determined as thesum of the numbers of positive wells for either of the two alleles. Instep 160, the proportion (P_(r)) of informative wells (an example of aparameter) contributed by the allele with more positive wells iscalculated.

P _(r)=No. of wells only positive for the allele with more positivewells/Total no. of wells positive for only one allele (A or G).

Other embodiments could use all wells with one of the alleles divided byall wells with at least one allele.

In step 170, it is determined whether the value of P_(r) shows anallelic imbalance. As accuracy and efficiency are desired, this task isnot straightforward. One method for determining an imbalance uses aBayesian-type likelihood method, sequential probability ratio testing(SPRT). SPRT is a method which allows two probabilistic hypotheses to becompared as data accumulate. In other words, it is a statistical methodto classify the results of digital PCR as being suggestive of thepresence or absence of allelic skewing. It has the advantage ofminimizing the number of wells to be analyzed to achieve a givenstatistical power and accuracy.

In an exemplary SPRT analysis, the experimental results would be testedagainst the null and alternative hypotheses. The alternative hypothesisis accepted when there is allelic ratio skewing in the sample. The nullhypothesis is accepted when there is no allelic ratio skewing in thesample. The value P_(r) would be compared with two cutoff values toaccept the null or alternative hypotheses. If neither hypothesis isaccepted, the sample would be marked as unclassified which means thatthe observed digital PCR result is not sufficient to classify the samplewith the desired statistical confidence.

The cutoff values for accepting the null or alternative hypotheses havetypically been calculated based on a fixed value of P_(r) under theassumptions stated in the hypotheses. In the null hypothesis, the sampleis assumed to exhibit no allelic ratio skewing. Therefore, theprobabilities of each well being positive for the A and the G alleleswould be the same and, hence, the expected value of P_(r) would be 1/2.In the alternative hypothesis, the expected value of P_(r) has beentaken to be 2/3 or about halfway between 0.5 and 2/3, e.g. 0.585. Also,due to a limited number of experiments, one can choose an upper bound(0.585+3/N) and a lower bound taken as (0.585-3/N).

B. Detection of Down Syndrome

In one embodiment of the present invention, digital SNP is used todetect fetal Down syndrome from a pregnant woman's plasma. Using markersspecific to fetal/placental cells, the ratio of alleles in chromosome 21may be measured. For example, to determine if an observed degree ofoverrepresentation of a PLAC4 allele is statistically significant, SPRTis used.

According to one exemplary embodiment, digital RNA-SNP determines animbalance in the ratio of polymorphic alleles of an A/G SNP, rs8130833,located on PLAC4 mRNA which is transcribed from chromosome 21 andexpressed by the placenta. For a heterozygous euploid fetus, the A and Galleles should be equally represented in the fetal genome (1:1 genomicratio); while in trisomy 21, the trisomic chromosome 21 would beassociated with an additional copy of one of the SNP alleles in thefetal genome giving a 2:1 ratio. The aim of the digital PCR analysis isto determine whether the amounts of the two PLAC4 alleles in theanalyzed sample are equal or otherwise. Thus, both the A and G PLAC4alleles are the target templates. A real-time PCR assay was designed toamplify PLAC4 mRNA and the two SNP alleles were discriminated by TaqManfluorescent probes. A schematic illustration of the analytical steps isshown in FIG. 2A.

FIG. 2A illustrates a digital RNA-SNP method 200 according to anembodiment of the present invention. In step 210, the sample isreceived. In step 220, the nucleic acid sequence, e.g. PLAC4 mRNA, isquantified in extracted RNA samples. In one embodiment, this is done byreal-time PCR for PLAC4 mRNA. In one aspect, this step provides theoperator with an idea about how much dilution is required before thetarget reaches the ‘realm’ of digital PCR analysis.

In step 230, the sample is diluted. In step 240, a concentration of thediluted sample is measured. The diluted sample concentration may beconfirmed to be ˜1 template/well (i.e. reference or non-referencesequence or either allele). Some embodiments use techniques described insection IV for this measurement. For example, we distributed the dilutedsample to 96 wells for real-time PCR analysis to confirm that a usabledilution has been achieved. The dilution concentration may also be leftas an unknown, thus removing this step as will be explained later.

In step 250, digital PCR is performed on each well of the array. Forexample, the same diluted sample was distributed to 384-wells forreal-time PCR analysis. From the PCR results, an amount of markers foreach nucleic acid sequence and the number of informative wells isidentified. An informative well is defined as one that is only positivefor the A or G allele but not both. In step 260, a calculation of anexpected value of P_(r) is performed. These steps will be described inmore detail later. The calculation includes determining a parameter fromvalues determined in step 250. For example, the actual average templateconcentration per well may be calculated.

In step 270, an SPRT or other likelihood-ratio test may be performed todetermine whether or not an imbalance exists. For a euploid case, weexpect an equal number of A-positive and G-positive wells. However, whentemplate molecules from a trisomy 21 fetus are analyzed, the number ofwells containing just one allele should be higher than that containingjust the other allele. In short, allelic imbalance is expected fortrisomy 21.

As mentioned above, SPRT is a Bayesian-type likelihood method whichallows two probabilistic hypotheses to be compared as data accumulate.In digital PCR analysis for trisomy 21 detection, the alternativehypothesis is accepted when allelic imbalance exists (i.e. trisomy 21detected); and the null hypothesis is accepted when there is no allelicimbalance (i.e. trisomy 21 not detected). The allele with the highernumber of counts is referred as the potentially overrepresented alleleand its proportion among all informative wells (P_(r)) would becalculated. SPRT is applied to determine if the P_(r) indicatessufficient degree of allelic imbalance that would be expected for atrisomy 21 sample.

Operationally, SPRT can be applied and interpreted through the use ofgraphs with a pair of SPRT curves that are constructed to define theprobabilistic boundaries for accepting or rejecting either hypothesis.FIG. 3 illustrates a graph having SPRT curves for determining Downsyndrome according to an embodiment of the present invention. The SPRTcurves plot the required proportion of informative wells positive forthe potentially overrepresented allele P_(r), (y-axis) for a given totalnumber of informative wells (x-axis) when confident classification couldbe made. As depicted in FIG. 3, the upper curve sets the probabilisticboundaries for accepting the alternative hypothesis; while the lowercurve sets the probabilistic boundaries for accepting the nullhypothesis.

The experimentally derived P_(r) value would be compared with theexpected value of P_(r) to accept or reject either hypothesis. If thenull hypothesis was accepted, the samples were classified as having beenobtained from pregnant women with euploid fetuses. If the alternativehypothesis was accepted, the samples were classified as having beenobtained from pregnant women with trisomy 21 fetuses. Alternatively,either hypothesis could not be accepted if the P_(r) for the givennumber of informative counts has not yet reached the required level ofstatistical confidence for disease classification. These cases weredeemed unclassifiable until more data were available. If diseaseclassification is not possible, additional 384-well plates may beperformed until the aggregated data become classifiable by SPRT.

SPRT thus offers the advantage that a smaller amount of testing isrequired for a given level of confidence than other statistical methods.In practical terms, SPRT allows the acceptance or rejection of either ofthe hypotheses as soon as the required amount of data has beenaccumulated and thus minimizes unnecessary additional analyses. Thisfeature is of particular relevance to the analysis of plasma nucleicacids which are generally present at low concentrations where the numberof available template molecules is limiting. In addition to a strictclassification, the classification may also include a percent accuracy.For example, a classification resulting from a comparison with a cutoffvalue may provide that a sample shows a likelihood of a nucleic acidsequence imbalance with a certain percentage, or equivalently thatdetermined imbalance is accurate to a certain percentage or other value.

A similar approach could be applied to determine the genotype of a fetuswith regard to either a mutation or genetic polymorphism, using fetalnucleic acids in maternal plasma or serum. One should recall that afetus would inherit half of its genome from its mother. As anillustration, consider a particular genetic locus with two alleles, Aand B. If the mother is a heterozygote with a genotype of AB, the fetuscould theoretically have a genotype of AA, BB or AB. If the fetus has agenotype of AB, i.e., the same as that of the mother, then there willonly be nucleic acids of the AB genotype (from both the mother andfetus) in maternal plasma. Thus, nucleic acid or allelic balance is seenin maternal plasma. On the other hand, if the fetus has a genotype of AAor BB, then there would be allelic imbalance with an overrepresentationof the A or the B allele, respectively, in maternal plasma. Thisconsideration is also applicable to disease-causing mutations (e.g.those causing cystic fibrosis or beta-thalassemia or spinal muscularatrophy), in which case A could be considered as the wildtype allele andB could be considered as the mutant allele.

II. Digital RCD

A disadvantage of digital RNA-SNP is that it can only be applied tocases heterozygous for the analyzed SNP. One improvement is that itwould be ideal if a noninvasive test for detecting fetal trisomy 21 orother fetal chromosomal aneuploidies (e.g. trisomy 18, 13, and the sexchromosome aneuploidies) based on circulating fetal nucleic acidanalysis were independent of the use of genetic polymorphisms. Thus, inone embodiment, chromosome dosage is determined by digital PCR analysisof a non-polymorphic chromosome 21 locus relative to one located on areference chromosome, namely chromosome 1 in this study. A change of theratio of chromosome 21 to chromosome 1 from 2:2 in the genome of aeuploid fetus is differentiated from a trisomy 21 case. In digital PCRanalysis for trisomy 21 detection, the two hypotheses to be comparedwould be the null hypothesis that there is no chromosomal imbalance(i.e. trisomy 21 not detected) and the alternative hypothesis that achromosomal imbalance exists (i.e. trisomy 21 detected).

This approach can be generalized to the other chromosomes involved inother chromosomal aneuploidies, e.g. chromosome 18 in trisomy 18,chromosome 13 in trisomy 13, chromosome X in Turner syndrome. Inaddition, apart from chromosome 1, other chromosomes not involved in theaneuploidies concerned can also be used as a reference chromosome. Asimilar approach can also be applied to the detection of cancer, byanalyzing the change of ratio of a chromosome commonly deleted, in part,in cancer, to a reference chromosome. Examples of the former includechromosome 5q in colorectal cancer, chromosome 3p in lung cancer andchromosome 9p in nasopharyngeal carcinoma. FIG. 2B lists some commoncancer-related chromosomal aberrations which result in sequenceimbalance.

FIG. 2A also illustrates a digital RCD method 205 according to anembodiment of the present invention. In one embodiment for steps220-230, extracted DNA is quantified, for example, via Nanodroptechniques and diluted to a concentration of approximately one targettemplate from either chromosomes 21 or the normalizing chromosome (suchas chromosome 1) per well. In one embodiment in step 240, theconfirmation may be performed by analyzing the diluted DNA sample by theassay using the chromosome 1 probe only in a 96-well format to confirmif ˜37% level of the wells were negative before proceeding to digitalRCD analysis using both TaqMan probes in 384-well plates. Thesignificance of the 37% will be described later in Section IV.

The testing of step 240 and results of step 250 may be done with areal-time PCR assay designed to amplify a paralogous sequence (Deutsch,S. et al. 2004 J Med Genet 41, 908-915) present on both chromosomeswhich are distinguished by paralogous sequence variations that arediscriminated by a pair of TaqMan probes. In this context, aninformative well is defined as one that is positive for either thechromosome 21 or chromosome 1 locus but not both. For a euploid fetus,the number of informative wells positive for either locus should beapproximately equal. For a trisomy 21 fetus, there should be anoverrepresentation of wells positive for chromosome 21 thanchromosome 1. The exact proportion of the overrepresentation isdescribed in the following sections.

III. Incorporating Percentage of Fetal Sequences

A disadvantage of embodiments of methods 200 and 205 described above isthat fetal specific markers are required. Accordingly, in one embodimentof the present invention, non-fetal specific markers are used. In orderto use such non-fetal specific markers, embodiments of the presentinvention measure the fractional concentration of fetal DNA in thematernal plasma (i.e. the biological sample). With such information, amore useful value of P_(r) may be calculated as follows.

Even with the small fractional percentage of fetal DNA in maternalplasma, a trisomy 21 fetus would contribute an additional dose ofchromosome 21 sequences per genome-equivalent (GE) of fetal DNA releasedinto maternal plasma. For example, a maternal plasma sample from aeuploid pregnancy containing 50 GE/ml of total DNA with 5 GE/ml DNAcontributed by the fetus (i.e. 10% fetal DNA fractional concentration)should contain a total of 100 copies (90 maternal copies+10 fetalcopies) of chromosome 21 sequences per milliliter of maternal plasma.For a trisomy 21 pregnancy, each fetal GE would contribute three copiesof chromosome 21, resulting in a total of 105 copies/ml (90 maternalcopies+15 fetal copies) of chromosome 21 sequences in maternal plasma.At 10% fetal DNA concentration, the amount of chromosome 21 derivedsequences in the maternal plasma of a trisomic pregnancy would thereforebe 1.05 times that of a euploid case. Thus, if an analytical approachcould be developed to determine this small degree of quantitativedifference, a polymorphism-independent test for noninvasive prenataldiagnosis of fetal trisomy 21 would be achieved.

Accordingly, the degree of overrepresentation would be dependent on thefractional fetal DNA concentration in the analyzed DNA sample. Forexample, when placental DNA is analyzed, the theoretical RCD ratio inthe fetal genome should be 3:2, i.e. 1.5-fold difference. However, asdescribed above, the theoretical RCD ratio would decrease to 1.05 when amaternal plasma sample containing 10% fetal DNA is analyzed. Theexperimentally derived P_(r) is calculated by dividing the number ofwells positive only for the chromosome 21 locus by the total number ofinformative wells. The experimentally derived P_(r) is subjected to theSPRT analysis with the calculated P_(r) and the theoretical RCD ratio.

FIG. 4 shows a method 400 of determining a disease state using apercentage of fetal nucleic acids according to an embodiment of thepresent invention. In step 410, the fractional percentage of fetalmaterial is measured. In one embodiment, the fractional percentage isdetermined by measuring the amount of a fetal-specific marker (e.g.Y-chromosome, genetic polymorphism markers (e.g. SNPs), placentalepigenetic signatures) in relation to a non-fetal-specific marker (i.e.gene sequence present in both mother and fetus). The actual measurementcould be done by real-time PCR, digital PCR, sequencing reactions(including massively parallel genomic sequencing) or any otherquantitative methods. In one aspect, it is preferable not to use thegene target that could potentially be in allelic imbalance for thismeasurement.

In step 420, digital PCR or other measurement method is performed,including diluting the sample, placing the diluted sample in the wells,and measuring the reactions in each well. In step 430, the PCR resultsare used to identify markers of different reference nucleic acidsequences (such as chromosomes or alleles). In step 440, the actualproportion (P_(r)) of the overrepresented sequence is calculated. Instep 450, the cutoff value for determining a disease state is calculatedusing the percentage of fetal material in the sample. In step 460, fromthe actual P_(r) and the cutoff value, it is determined whether animbalance exists.

In one embodiment, the fractional percentage of reference nucleic acidsequences is incorporated in a digital RNA-SNP method. Thus, wheninvestigating a LOH due to cancer cells, this can be done with tumorsamples with less than 50% cancer cells. It also may be used on sampleswith greater than 50% cancer cells to obtain a more accurate P_(r) andthus reduce the number of false positives, which would lead to incorrectdiagnoses. In another embodiment, the fetal nucleic acid percentage isincorporated in a digital PCR method to determine if a fetus hasinherited a parental gene mutation (e.g. that causing cystic fibrosis orbeta-thalassemia or spinal muscular atrophy) or polymorphism frommaternal plasma nucleic acid analysis.

IV. Incorporating Average Concentration Per Well

Another disadvantage of previous methods (e.g. Zhou, W. et al. 2002,supra) is that the average concentration of templates per well (m) isrequired to be 1 per well. Given that it is difficult to obtain an exactconcentration, this can lead to inaccuracies. Furthermore, even with anexact concentration of 1 template per well, previous methods haveignored the statistical distribution of the templates in a well. In theprevious methods, i.e. the old algorithm, the expected value of P_(r)for accepting the alternative hypothesis is assumed to be the allelicratio and, thus, is independent of the average concentration of thetemplate DNA per well.

However, due to a natural statistical variation of the templates in thediluted sample, there will not be exactly 1 template per well.Embodiments of the present invention measure the average concentrationof at least one of the sequences, which is then used to calculate thecutoff value, i.e. the expected P_(r). In one aspect, this calculationinvolves a statistical distribution to determine a probability of a wellcontaining the different nucleic acid sequences, which is then used todetermine the expected P_(r).

In one embodiment, the average concentration is taken of one referencenucleic acid sequence, which in one instance is nucleic acid sequencewith the lower concentration in the DNA sample. In the case of a samplewithout an imbalance, the concentrations of the two sequences in thesample would be the same and either one can be regarded as the referenceallele. In the case of a sample with, for example, LOH, the allele whichis deleted in the cancer cells would be regarded as the referenceallele. The average concentration of the reference allele would bedenoted as m_(r). In another embodiment, the sequence with the higherconcentration may be taken as the reference sequence.

A. Digital-SNP. Example Using SPRT and Digital PCR

FIG. 5 shows a method 500 of determining a disease state using anaverage template concentration according to an embodiment of the presentinvention. In step 510, an amount of the different sequences aremeasured. This may be done for example by counting the makers in adigital PCR experiment as explained above. However, it may be done byother methods that do not involve an amplification step or that do notuse a fluorescent marker, but could use other properties, such asphysical properties like mass, specific optical properties orbase-pairing properties.

In step 520, the actual proportion of the overrepresented sequence isdetermined. This may be done as described above by taking the number ofwells showing only that sequence and dividing by the number ofinformative wells. In step 530, the average concentration of at leastone of the sequences is measured (the reference sequence). In oneembodiment, the reference sequence is the overrepresented sequence. Inanother embodiment, the reference sequence is the underrepresentedsequence. The measurement may be done by counting the number of wellsnegative for the reference sequence in the digital PCR experiment. Therelationship between the proportion of negative wells and the averagetemplate concentration is described by the Poisson distribution asdescribed in the next subsection.

In step 540, an expected amount of wells positive for the differentsequences is calculated, for example, using a Poisson distribution. Theexpected amount may be as a probability of the sequence per well, anaverage sequence per well, the number of wells containing the sequenceor any other suitable quantity. In step 550, the expected P_(r) iscalculated from the expected amounts. In step 560, a cutoff value iscalculated from the expected P_(r), for example, by using SPRT. In step570, a classification of the nucleic acid sequence imbalance isdetermined. Specific aspects of method 500 are now described.

1. Determining Expected Amount of Sequences

Once the average concentration per well (reaction or reaction mixture)is known from step 530, the expected number of wells showing thatsequence may be calculated in step 540. This amount may be expressed asa %, a fractional value, or an integer value. Using a specific examplefor illustration, assume the average concentration of the referencetemplate per well (m_(r)) is 0.5 per well and the genotype of thetrisomy 21 fetus at the PLAC4 SNP, rs8130833, is AGG. Therefore, thereference template would be the A allele and the overrepresentedtemplate would be the G allele.

In one embodiment, a Poisson distribution is assumed for thedistribution of the A allele among the reaction mixtures of the wells ofthe measurement procedure, such as digital PCR. In other embodiments,other distribution functions are used, such as binomial distribution.

The Poisson equation is:

${P(n)} = \frac{m^{n}e^{- m}}{n!}$

where, n=number of template molecules per well; P(n)=probability of ntemplate molecules in a particular well; and m=average number oftemplate molecules in one well in a particular digital PCR experiment.

Accordingly, the probability of any well not containing any molecule ofthe A allele at an average A-allele concentration of 0.5 would be:

${P(0)} = {\frac{0.5^{0}e^{- 0.5}}{0!} = {e^{- 0.5} = {0.6065.}}}$

Hence, the probability of any well containing at least one molecule ofthe A allele would be: 1−0.6065=0.3935. Therefore, ˜39% of the wellswould be expected to contain at least one molecule of the A allele.

As for the non-reference nucleic acid sequence, for each cell of atrisomy 21 fetus, the genomic ratio of A to G would be 1:2. Assumingthat the A to G ratio in the extracted RNA or DNA sample would remainunchanged, the average concentration of the G allele per well would betwo times that of the A allele, i.e. 2×0.5=1.

Accordingly, the probability of any well not containing any molecule ofthe G allele at an average G-allele concentration of 1 would be:

${P(0)} = {\frac{1^{0}e^{- 1}}{0!} = {e^{- 1} = 0.3679}}$

Hence, the probability of any well containing at least one molecule ofthe G allele would be: 1−0.3679=0.6321. Therefore, ˜63% of the wellswould be expected to contain at least one molecule of the G allele.

2. Determining Proportion of Overrepresented Sequence

After the expected amounts are calculated, the proportion of theoverrepresented nucleic acid sequence may be determined. Assuming thatthe filling of the wells with the A allele and the G allele areindependent, the probability of a well containing both alleles would be0.3935×0.6321=0.2487. Therefore, ˜25% of the wells would be expected tocontain both alleles.

The proportion of wells expected to contain the A allele but not the Gallele would be the number of wells containing at least one A allelededucted by the number of wells containing both the A and G alleles:0.3935−0.2487=0.1448. Similarly, the proportion of wells expected tocontain the G allele but not the A allele would be:0.6321−0.2487=0.3834. An informative well is defined as a well beingpositive for either the A allele or the G allele but not both.

Hence, the expected ratio of wells containing the A allele relative tothe G allele in a digital RNA-SNP analysis is 0.1448/0.3834. In otherwords, the proportion of wells positive only for the G allele is 2.65times that of wells positive only for the A allele. This is in contrastto the fetal genomic ratio where the overrepresented allele is 2 timesthat of the other allele.

For SPRT analysis, the proportion of the informative wells positive forthe overrepresented allele (P_(r)) is calculated and interpreted usingSPRT curves. In the current example, the proportion of informative wellswould be: 0.1448+0.3834=0.5282. Hence, the expected Pr of a trisomy 21case at m_(r) 0.5 is: 0.3834/0.5282=0.73.

As the average template concentration (m) is a key parameter in thePoisson equation, the P_(r) would vary with m. FIG. 6 shows a table 600that tabulates the expected digital RNA-SNP allelic ratio and P_(r) oftrisomy 21 samples for a range of template concentrations expressed asthe average reference template concentration per well (m_(r)) accordingto an embodiment of the present invention. Table 600 shows the expectedallelic ratio and proportion of informative wells positive for theoverrepresented allele (P_(r)) for a series of average referencetemplate concentrations per well (m_(r)).

The expected value of P_(r) varies with the average concentration of thereference allele per well (m_(r)) in a non-linear fashion. As shown intable 600, the expected value of P_(r) for accepting the alternativehypothesis would increase with m_(r). As the expected value of P_(r) foraccepting the null hypothesis is fixed at 0.5, the samples with andwithout allelic imbalance would separate more widely in terms of thevalue of P_(r) when m_(r) increases. Note that in other embodiments thevalue for accepting the null hypothesis may be other than 0.5. Thismight occur when the normal ratio is different than 1:1, e.g., 5:3, andthus an imbalance would occur when the ratio deviates from 5:3. Thedifference in the amounts of the two different nucleic acid sequenceswould then be determined on a case by case basis.

However, as previous methods (e.g., Zhou, W. et al. 2002, supra) used afixed expected value of P_(r) for LOH samples, they underestimated thevalue of P_(r) for those samples with LOH (alternative hypothesisaccepted). The degree of underestimation would increase with m_(r). Inother words, the higher the average concentration of the referenceallele in the DNA sample, the more inaccurate the old methods would be.This underestimation of P_(r) for accepting the alternative hypothesiswould lead to the inaccurate calculation of the cutoff values foraccepting both the null and alternative hypotheses.

3. Calculating Cutoff Values Based on Expected P_(r)

For embodiments using SPRT, one may use the equations for calculatingthe upper and lower boundaries of the SPRT curves from El Karoui at al.(2006). Furthermore, the level of statistical confidence preferred foraccepting the null or alternative hypothesis could be varied throughadjusting the threshold likelihood ratio in the equations. In thisstudy, a threshold likelihood ratio of 8 is used because this value hadbeen shown to provide satisfactory performance to discriminate sampleswith and without allelic imbalance in the context of cancer detection.Thus, in one embodiment, the equations for calculating the upper andlower boundaries of the SPRT curves are:

Upper boundary=[(ln 8)/N−ln δ]/ln γ

Lower boundary=[(ln 1/8)/N−ln δ]/ln γ

where, δ=(1−θ₁)/(1−θ₀)

-   -   γ=−(θ₁(1−θ₀)/θ₀(1−θ₁)    -   θ₀=proportion of informative wells containing the non-reference        allele if the null hypothesis is true        -   =0.5 (see below)    -   θ₁=proportion of informative wells containing the non-reference        (i.e. overrepresented) allele if the alternative hypothesis is        true    -   N=number of informative wells        -   =number of wells positive for either allele only    -   (ln is a mathematical symbol representing the natural logarithm,        i.e. log_(e).)

For the determination of θ₀ for accepting the null hypothesis, thesample is assumed to be obtained from a pregnant woman carrying aeuploid fetus. Under this assumption, the expected number of wellspositive for either template would be 1:1, and thus the expectedproportion of informative wells containing the non-reference allelewould be 0.5.

For the determination of θ₁ for accepting the alternative hypothesis,the sample is assumed to be obtained from a pregnant woman carrying atrisomy 21 fetus. The calculations for the expected P_(r) of trisomy 21cases for digital RNA-SNP analysis are detailed in Table 600. Hence, θ₁for digital RNA-SNP analysis refers to the data shown in the last columnof table 600.

4. Measurement of Average Concentration

The measurement of m_(r) may be performed through a variety ofmechanisms as known or will be known to one skilled in the art. In oneembodiment, the value of m_(r) is determined during the experimentalprocess of digital PCR analysis. As the relationship between the valueof m_(r) and the total number of wells being positive for the referenceallele can be governed by a distribution (e.g. the Poissondistribution), m_(r) can be calculated from the number of wells beingpositive for the reference allele using this formula:

m _(r)=−ln(1−proportion of wells being positive for the referenceallele)

Note that ln is the natural logarithm, i.e., log_(e). This approachprovides a direct and precise estimation of m_(r) in the DNA sample usedfor the digital PCR experiment.

This method may be used to achieve a desired concentration. For example,the extracted nucleic acids of a sample may be diluted to a specificconcentration, such as one template molecule per reaction well, as donein step 240 of method 200. In an embodiment using the Poissondistribution, the expected proportion of wells with no template may becalculated as e^(−m), where m is the average concentration of templatemolecules per well. For example, at an average concentration of onetemplate molecule per well, the expected proportion of wells with notemplate molecule is given by e⁻¹, i.e., 0.37 (37%). The remaining 63%of wells will contain one or more template molecules. Typically, thenumber of positive and informative wells in a digital PCR run would thenbe counted. The definition of informative wells and the manner by whichthe digital PCR data are interpreted depends on the application.

In other embodiments, the average concentration per well, m_(r), ismeasured by another quantification method, for example, quantitativereal-time PCR, semi-quantitative competitive PCR, real-competitive PCRusing mass spectrometric methods, etc.

B. Digital RCD

Digital RCD using the average concentration may be performed in asimilar manner to the digital SNP method described above. The numbers ofwells positive for the reference chromosome (non-chromosome 21) marker,the chromosome 21 marker and both markers can be determined by digitalPCR. The average concentration of the reference marker per well (m_(r))can be calculated from the total number of wells negative for thereference marker, irrespective of the positivity of the chromosome 21marker, according to the Poisson probability function as in thecalculation of m_(r) for the digital SNP analysis.

SPRT analysis may then be used for classifying a plasma sample as beingobtained from a pregnant woman carrying a euploid or a trisomy 21 fetus.The null hypothesis would be accepted when the fetus was euploid. Inthis scenario, the expected ratio for the wells positive for thereference marker and chromosome 21 marker would be 1:1 and, thus, theexpected proportion of informative wells with positive signal forchromosome 21 marker would be 0.5. The alternative hypothesis would beaccepted when the fetus was trisomic for chromosome 21. In thisscenario, if the sample DNA was solely derived from the fetus, theaverage concentration of the chromosome 21 marker in each well would be3/2 times the average concentration of the reference marker (m_(r)).

While digital RCD may be used to determine chromosome dosage through thedetection of fetal-specific markers, e.g. epigenetic signatures of theplacenta (Chim, S S C. et al. 2005 Proc Natl Acad Sci USA 102,14753-14758), an embodiment of the digital RCD analysis usesnon-fetal-specific markers. Thus, an additional step of measuring thepercentage of fetal material would occur when non-fetal specific markersare used. Therefore, the average concentration of the chromosome 21marker per well would be dependent on the proportion of the fetal DNA inthe sample and can be calculated using: m_(r) [(200%+fetal DNApercentage)/200%].

Again using a specific example for illustration, the averageconcentration of the reference template, chromosome 1, per well (m_(r))is assumed to be 0.5 and 50% of the DNA is assumed to be derived fromthe fetus and 50% of the DNA in the sample is derived from the mother.

Accordingly, using the Poisson distribution, the probability of any wellnot containing any molecule of the chromosome 1 locus when its averageconcentration is 0.5 per well would be:

${P(0)} = {\frac{0.5^{0}e^{- 0.5}}{0!} = {e^{- 0.5} = 0.6065}}$

Hence, the probability of any well containing at least one molecule ofthe chromosome 1 locus would be: 1−0.6065=0.3935. Therefore, ˜39% of thewells would be expected to contain at least one molecule of the locus.

For each cell of this trisomy 21 fetus, the genomic ratio of chromosome21 to chromosome 1 would be 3:2. The ratio between chromosome 21 andchromosome 1 in the DNA sample would be dependent on the fractionalfetal DNA concentration (fetal DNA %) and would be: 3×fetal DNA %+2(1−fetal DNA %): 2×fetal DNA %+2×(1−fetal DNA %). Thus, in this casewhen the fractional fetal DNA concentration is 50%, the ratio would be:(3×50%+2×50%)/(2×50%+2×50%)=1.25. If the digital SNP method did not usefetal specific markers, such a calculation could also be used tocalculate the average concentration of the non-reference sequence.

Hence, when the average concentration of the chromosome 1 locus per wellis 0.5, the average concentration of the chromosome 21 locus per wellis: 1.25×0.5=0.625. Accordingly, the probability of any well notcontaining any molecule of the chromosome 21 locus when its averageconcentration is 0.625 per well would be:

${P(0)} = {\frac{0.625^{0}e^{- 0.625}}{0!} = {e^{- 0.625} = 0.5353}}$

Hence, the probability of any well containing at least one molecule ofthe chromosome 21 locus would be: 1−0.5353=0.4647. Therefore, ˜46% ofthe wells would be expected to contain at least one molecule of thelocus. Assuming that the filling of the wells with either loci areindependent, the probability of a well containing both loci would be0.3935×0.4647=0.1829. Therefore, ˜18% of the wells would be expected tocontain both loci.

The proportion of wells expected to contain the chromosome 1 locus butnot the chromosome 21 locus would be the number of wells containing atleast one chromosome 1 locus deducted by the number of wells containingboth loci: 0.3935−0.1829=0.2106. Similarly, the proportion of wellsexpected to contain the chromosome 21 locus but not both loci would be:0.4647−0.1829=0.2818. An informative well is defined as a well positivefor either the chromosome 1 locus or the chromosome 21 locus but notboth.

Hence, the expected chromosome 21 to chromosome 1 ratio in digital RCDanalysis is 0.2818/0.2106=1.34. In other words, the proportion of wellspositive only for the chromosome 21 locus is 1.34 times that of wellspositive only for the chromosome 1 locus. This is in contrast to theratio of 1.25 in the DNA sample.

For SPRT analysis, the proportion of the informative wells positive forthe chromosome 21 locus (P_(r)) would need to be calculated andinterpreted using SPRT curves. In the current example, the proportion ofinformative wells would be: 0.2106+0.2818=0.4924. Hence, the expectedP_(r) of a trisomy 21 case with 50% fetal DNA at m_(r) 0.5 is:0.2818/0.4924=0.57.

As the average template concentration (m) is a key parameter in thePoisson equation, the P_(r) would vary with m. FIG. 7 shows a table 700that tabulates the expected P_(r) for the fractional fetal DNAconcentrations of 10%, 25%, 50% and 100% in trisomy 21 samples at arange of template concentrations expressed as the average referencetemplate concentration per well (m_(r)) according to an embodiment ofthe present invention. The calculations for the expected P_(r) oftrisomy 21 cases for digital RCD analyses are detailed in table 700.Hence, θ₁ for digital RCD analysis of samples with varying fetal DNAfractional concentrations can be obtained from the columns showing thecorresponding expected P_(r)-values in table 700.

C. Results

1. Comparison of Different m_(r)

The basis for the difference between the theoretical (as in the fetalgenome) and experimentally-expected degree of allelic or chromosomalimbalance and the calculations to determine the latter for a range ofm_(r) values are shown in tables 600 and 700. In digital RNA-SNPanalysis of a trisomy 21 sample, when m_(r)=0.5, wells containing justthe overrepresented allele with respect to wells containing just thereference allele, namely the digital RNA-SNP ratio, is 2.65 (table 600).In digital RCD analysis of a specimen composed of 100% fetal DNA, whenm_(r)=0.5, wells positive solely for the chromosome 21 locus withrespect to those positive solely for the chromosome 1 locus, namely thedigital RCD ratio, is 1.7 (table 700) (P_(r)=0.63, thus the digital RCDratio is 0.63/(1−0.63)=1.7). As the fractional fetal DNA concentrationdecreases, the digital RCD ratio decreases for the same m_(r) (table700).

As shown in tables 600 and 700, the extent of allelic or chromosomaloverrepresentation increases with m_(r). However, the percentage ofinformative wells approaches its maximum around m_(r)=0.5 and decreasesgradually with further increase in m_(r). In practice, the decline inthe proportion of informative wells could be compensated by increasingthe total number of wells analyzed if the amount of template moleculefor the specimen is not limiting, but additional wells would require anincrease in reagent costs. Hence, optimal digital PCR performance is atrade-off between the template concentration and total number of wellstested per sample.

2. Example Using SPRT Curves

As discussed above, the expected degree of allelic or chromosomalimbalance for a digital PCR experiment is dependent on the actualtemplate concentration per reaction mixture (e.g. a well). We describethe template concentration based on the reference allele, i.e. theaverage reference template concentration per well (m_(r)). As shown inthe above equation, the expected P_(r) can be used to determine theplotting of the upper and the lower SPRT curves. Since the expectedP_(r) is in turn dependent on the value of m_(r), the plotting of theSPRT curves would essentially be dependent on the value of m_(r). Thus,in practice, a set of SPRT curves relevant for the actual m_(r) of adigital PCR dataset would need to be used for the interpretation of theP_(r) from that particular run.

FIG. 8 shows a plot 800 illustrating the degree of differences in theSPRT curves for m_(r) values of 0.1, 0.5 and 1.0 for digital RNA-SNPanalysis according to an embodiment of the present invention. Each setof digital PCR data should be interpreted with the specific curvesrelevant to the exact m_(r) value of that particular run. Note thatsince the expected degree of allelic or chromosomal imbalance for thedigital RNA-SNP and RCD approaches are different (2:1 for the former and3:2 for the latter), different sets of SPRT curves are needed for thetwo digital PCR systems. The experimentally derived P_(r) is interpretedwith the relevant SPRT curves selected by the corresponding m_(r) of thedigital PCR run. This is in contrast to the previous reported use ofSPRT for molecular detection of LOH by digital PCR where a fixed set ofcurves had been used.

The practical manner for interpreting the digital PCR data using SPRT isillustrated below using a hypothetical digital RNA-SNP run. Afterdigital RNA-SNP analysis of each case, the number of wells positive forthe A allele only, the G allele only or both alleles are counted. Thereference allele is defined as the allele with the smaller number ofpositive wells. The value of m_(r) is calculated using the total numberof wells negative for the reference allele, irrespective of whether theother allele is positive, according to the Poisson probability densityfunction. The data of our hypothetical example are as follows:

In a 96-well reaction, 20 wells are positive for the A allele only, 24wells are positive for the G allele only, and 33 wells are positive forboth alleles. The A allele is regarded as the reference allele becausethere are less A-positive than G-positive wells. The number of wellsnegative for the reference allele is 96−20−33=43. Therefore, m_(r) canbe calculated using the Poisson equation: −ln(43/96)=0.80. Theexperimentally determined P_(r) of this case is: 24/(20+24)=0.55.

According to table 600, the expected P_(r) of a trisomy 21 sample atm_(r)=0.8 is 0.76. Thus, θ₁ is 0.76 for this case. The SPRT curves basedon θ₁=0.76 would be used to interpret the experimentally derived P_(r)of this case which is 0.55. When P_(r)=0.55 is fitted onto the relevantSPRT curves, the data point falls under the lower curve. Hence, the caseis classified as euploid, see FIG. 3.

3. Comparison to Old Method

FIG. 9A shows a table 900 of a comparison of the effectiveness of thenew and old SPRT algorithms for classifying euploid and trisomy 21 casesin 96-well digital RNA-SNP analyses. FIG. 9B shows a table 950 of acomparison of the effectiveness of the new and old SPRT algorithms forclassifying euploid and trisomy 21 cases in 384-well digital RNA-SNPanalyses. The new algorithm refers to the selection of SPRT curvesspecific for the m_(r) derived from the digital PCR data. The oldalgorithm refers to the use of a fixed set of SPRT curves for alldigital PCR runs. The effect of incorrect calculation of the cutoffvalues on the accuracy of classification is revealed by the simulationanalysis shown in table 900.

Compared with the use of a fixed set of SPRT curves in previous studies,the proportion of unclassifiable data is much lower with our approach,as shown in tables 900 and 950. For example, using our approach, atm_(r)=0.5, 14% and 0% of trisomy 21 samples would be unclassifiable for96-well and 384-well digital RNA-SNP analyses, respectively, but 62% and10%, respectively, with the use of fixed curves (Table 900). Hence, ourapproach allows disease classification with lesser number of informativewells.

As shown in table 900, the new algorithm is more accurate in classifyingthe samples as having or not having allelic ratio skewing for all valuesof m_(r) from 0.1 to 2.0. For example, when m_(r) equals 1.0 and a96-well digital RNA-SNP run is performed, the new algorithm correctlyclassifies 88% and 92% of samples with and without allelic ratioskewing, respectively, whereas the percentage of correct classificationof samples with and without allelic ratio skewing using the oldalgorithm is only 19% and 36%, respectively.

Using the new algorithm, the separation of samples with and withoutallelic ratio skewing would increase with m_(r). As a result, theclassification accuracies would increase with m_(r). The effect ofincrease in separation of the two groups of samples on theclassification accuracy would diminish when m_(r) increases to beyond2.0 because of the reduction in the percentage of informative wells. Incontrast, using the old algorithm, the classification accuraciessignificantly reduce when m_(r) increases because of the increaseddeviation of expected P value from its true value.

Our experimental and simulation data show that digital RNA-SNP is aneffective and accurate method for trisomy 21 detection. As PLAC4 mRNA inmaternal plasma is derived purely from the fetus, for 12 of the 13maternal plasma samples tested, only one 384-well digital PCR experimentwas required for correct classification (Table 1350 of FIG. 13B). Thishomogenous, real-time digital PCR-based approach thus offers analternative to the mass spectrometry-based approach for RNA-SNP analysis(Lo, Y M D, et al. 2007 Nat Med, supra). Apart from placental-specificmRNA transcripts, we also envision that other types of fetal-specificnucleic acid species in maternal plasma could be used for digitalPCR-based detection of fetal chromosomal aneuploidies. One example isfetal epigenetic markers (Chim, S S C et al. (2005) Proc Natl Acad SciUSA 102, 14753-14758; Chan, K C A et al. (2006) Clin Chem 52,2211-2218), which have recently been used for the noninvasive prenataldetection of trisomy 18 using the epigenetic allelic ratio (EAR)approach (Tong, Y K et al. (2006) Clin Chem 52, 2194-2202). Thus, wepredict that digital EAR would be a possible analytical technique.

V. Increasing %, Multiple Markers, and PCR Alternatives

As described above, the application of embodiments of the presentinvention to DNA extracted from maternal plasma can be complicated whenthe fetal DNA only constitutes a minor fraction of maternal plasma DNA,with a mean fractional concentration of some 3% between weeks 11 and 17of gestation. Nevertheless, as shown herein, digital RCD allowsaneuploidy detection even when the aneuploid DNA is present as a minorpopulation. With a decreasing fractional concentration of fetal DNA,such as may be present during early gestation, a larger number ofinformative counts is needed for digital RCD. A significance of thepresent work, as summarized in table 1200 of FIG. 12, is that we haveprovided a set of benchmark parameters, e.g. fractional fetal DNA andtotal template molecules required, which diagnostic assays can be builtupon. In our opinion, 7680 total number of reactions for a fractionalfetal DNA concentration of 25% is a particularly attractive set ofbenchmark parameters. These parameters should allow euploid and trisomy21 samples to be classifiable correctly 97% of the time, as shown intable 1200.

The number of plasma DNA molecules that are present per unit volume ofmaternal plasma is limited (Lo, Y M D. et al. 1998 Am J Hum Genet 62,768-7758). For example, in early pregnancy, the median maternal plasmaconcentration of an autosomal locus, the β-globin gene, has been shownto be 986 copies/mL, with contributions from both the fetus and mother(Lo, Y M D. et al. 1998 Am J Hum Genet 62, 768-7758). To capture 7,680molecules, DNA extracted from some 8 mL of maternal plasma would beneeded. This volume of plasma, obtainable from ˜15 mL of maternal blood,is at the limit of routine practice. However, we envision that multiplesets of chr21 and reference chromosome targets can be combined fordigital RCD analysis. For 5 pairs of chr21 and reference chromosometargets, just 1.6 mL of maternal plasma would be needed to provide thenumber of template molecules needed for analysis. Multiplex singlemolecule PCR could be performed. The robustness of such multiplex singlemolecule analysis has been demonstrated previously for single moleculehaplotyping (Ding, C. and Cantor, C R. 2003 Proc Natl Acad Sci USA 100,7449-7453).

Alternatively, to achieve a fractional fetal DNA concentration of 25%,methods may allow the selective enrichment of fetal DNA (Li, Y. et al.2004 Clin Chem 50, 1002-1011) or the suppression of the maternal DNAbackground (Dhallan, R et al. 2004 JAMA 291, 1114-1119) or both, inmaternal plasma. Apart from such physical methods for fetal DNAenrichment and maternal DNA suppression, it would also be possible touse a molecular enrichment strategy, such as by targeting fetal DNAmolecules which exhibit a particular DNA methylation pattern (Chim, S SC et al, 2005 Proc Natl Acad Sci USA 102, 14753-14758, Chan, K C A etal. 2006 Clin Chem 52, 2211-2218; Chiu, R W K et al. 2007 Am J Pathol170, 941-950.)

Additionally, there are now a number of alternative approaches to themanual set up of digital real-time PCR analyses as used in the currentstudy for conducting digital PCR. These alternative approaches includemicrofluidics digital PCR chips (Warren, L et al. 2006 Proc Natl AcadSci USA 103, 17807-17812; Ottesen, E A et al. 2006 Science 314,1464-1467), emulsion PCR (Dressman, D et al. 2003 Proc Natl Acad Sci USA100, 8817-8822), and massively parallel genomic sequencing (Margulies,M. et al. 2005 Nature 437, 376-380) using for example the Roche 454platform, the Illumina Solexa platform, and the SOLiD™ system of AppliedBiosystems, etc. With regard to the latter, our method is alsoapplicable to massively parallel sequencing methods on single DNAmolecules, which do not require an amplification step, such as theHelicos True Single Molecule DNA sequencing technology (Harris T D etal. 2008 Science, 320, 106-109), the single molecule, real-time (SMRT™)technology of Pacific Biosciences, and nanopore sequencing (Soni G V andMeller A. 2007 Clin Chem 53, 1996-2001). With the use of these methods,digital RNA-SNP and digital RCD could be performed rapidly on a largenumber of samples, thus enhancing the clinical feasibility of themethods proposed here for noninvasive prenatal diagnosis.

Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

I. Computer Simulations

Computer simulation was performed to estimate the accuracy of diagnosingtrisomy 21 using the SPRT approach. The computer simulation wasperformed with the Microsoft Excel 2003 software (Microsoft Corp., USA)and SAS 9.1 for Windows software (SAS Institute Inc., NC, USA). Theperformance of digital PCR is an interplay between the referencetemplate concentration (m_(r)), number of informative counts andprojected degree of allelic or chromosomal imbalance (P_(r)). Separatesimulations were performed for a range of each of these variables. Sincethe decision boundaries of the SPRT curves for digital RNA-SNP anddigital RCD were different, the simulation analyses for the two systemswere performed separately.

For each digital PCR condition simulated (i.e. m_(r), fetal DNAfractional concentration, total well number), two rounds of simulationwere performed. The first round simulated the scenario that the testedsamples were obtained from pregnant women carrying euploid fetuses. Thesecond round simulated the scenario when the tested samples wereobtained from pregnant women carrying trisomy 21 fetuses. For eachround, testing of 5000 fetuses was simulated.

A. RNA-SNP

For digital RNA-SNP, simulations of a 384-well experiment with m_(r)=0.1to m_(r)=2.0 were performed. At each m_(r) value, we simulated thescenario whereby 5000 euploid and 5000 trisomy 21 fetuses were tested.The SPRT curves appropriate for the given m_(r) were used to classifythe 10,000 fetuses. FIG. 10 is a table 1000 showing the percentages offetuses correctly and incorrectly classified as euploid or aneuploid andthose not classifiable for the given informative counts according to anembodiment of the present invention. The accuracies for diagnosingeuploid and aneuploid cases are both 100%, for m_(r) between 0.5 and2.0. When m_(r)=0.1, only 57% and 88% of euploid and trisomy 21 fetusescould be accurately classified after the analysis of 384 wells.

The simulation data were generated as described in the following steps:

In step 1, for each well, two random numbers were generated using theRandom(Poisson) function of the SAS program(www.sas.com/technologies/analytics/statistics/index.html) to representthe A and the G alleles, respectively. The Random(Poisson) functionwould generate positive integers starting from 0 (i.e. 0, 1, 2, 3, . . .) and the probability of each integer being generated was dependent onthe probability of this number according to the Poisson probabilitydensity function for a given mean value which represented the averageconcentration of the alleles per well. A well was regarded as positivefor the A allele if the random number representing the A allele waslarger than zero, i.e. contained 1 or more molecules of the A allele.Similarly, the well was regarded as positive for the G allele if therandom number representing the G allele was larger than zero.

To simulate the scenario of a pregnant woman carrying a euploid fetus,the same mean value was used for generating the random numbers for the Aallele and the G allele. For example, in the analysis simulating digitalRNA-SNP analyses at m_(r)=0.5, the mean value for either the A allele ofthe G allele was set identically to 0.5 which meant an averageconcentration for either allele of 0.5 molecule per well. Using thePoisson equation, at a mean concentration of 0.5, the proportion ofwells being positive for the A or the G alleles would be the same andwas 0.3935, see table 600.

When simulating the digital RNA-SNP analysis of a pregnant woman with atrisomy 21 fetus at m_(r)=0.5, the average concentration of theoverrepresented allele per well would be expected to be 2 times of thatof the reference allele, i.e. 1. In this situation, the probability of awell being positive for the overrepresented allele was 0.6321, see table600.

After generating a random number for a digital PCR well, the well couldbe classified as one of the following statuses:

a. negative for both the A and the G alleles

b. positive for both the A and the G alleles

c. positive for the A allele but negative for the G allele

d. positive for the G allele but negative for the A allele

In step 2, step 1 was repeated until the desired number of wells, 384wells for the current simulation, had been generated. The numbers ofwells positive for the A allele only and the G allele only were counted.The allele with less positive wells was regarded as the reference alleleand the allele with more positive wells was regarded as the potentiallyoverrepresented allele. The number of the informative wells was thetotal number of wells positive for either allele but not both. Theproportion of informative wells containing the potentiallyoverrepresented allele (P_(r)) was then calculated. The upper and lowerboundaries for the relevant SPRT curves to accept the null oralternative hypothesis were calculated according to an embodiment of thepresent invention.

In step 3, 5000 simulations were performed for each of the two scenariosof the pregnant woman carrying a euploid or a trisomy 21 fetus. Eachsimulation could be regarded as an independent biological sampleobtained from pregnant women. In Table 1000, the correct classificationof euploid cases refers to those euploid cases in which the nullhypothesis was accepted and the incorrect classification of euploidcases refers to those euploid cases in which the alternative hypothesiswas accepted. Similarly, those trisomy 21 cases in which the alternativehypothesis was accepted were regarded as correctly classified and thosetrisomy 21 cases in which the null hypothesis was accepted were regardedas incorrectly classified. For both groups, those cases in which neitherthe null or alternative hypothesis was accepted after the pre-specifiedtotal number of wells had been simulated were regarded as unclassified.

In step 4, steps 1 to 3 were performed for m_(r) ranging from 0.1 to 2.0at increments of 0.1.

B. RCD

FIG. 11 is a table 1100 showing computer simulations for digital RCDanalysis for a pure (100%) fetal DNA sample for m_(r) ranging from 0.1to 2.0 according to an embodiment of the present invention. As thefractional fetal DNA concentration becomes lower, the degree ofchromosome 21 overrepresentation diminishes and thus a larger number ofinformative wells for accurate disease classification is required.Hence, simulations were further performed for fetal DNA concentrationsof 50%, 25% and 10% for a total well number ranging from 384 to 7680wells at m_(r)=0.5.

FIG. 12 is a table 1200 showing results of computer simulation ofaccuracies of digital RCD analysis at m_(r)=0.5 for the classificationof samples from euploid or trisomy 21 fetuses with different fractionalconcentrations of fetal DNA according to an embodiment of the presentinvention. The performance of digital RCD is better for cases with ahigher fetal DNA fractional concentration. At 25% fetal DNAconcentration and a total number of 7680 PCR analyses, 97% of botheuploid and aneuploid cases would be classifiable with no incorrectclassification. The remaining 3% of cases require further analyses untilclassification can be achieved.

The procedures for simulating digital RCD analyses were similar to thosedescribed for digital RNA-SNP analysis. The steps for the simulationsare described below:

In step 1, two random numbers under the Poisson probability densityfunction were generated to represent the reference locus, chromosome 1,and the chromosome 21 locus. For subjects carrying euploid fetuses, theaverage concentrations of both the chromosome 1 and chromosome 21 lociwere the same. In this simulation analysis, an average templateconcentration of 0.5 for each locus per well was used. For subjectscarrying trisomy 21 fetuses, the m_(r) in this simulation was 0.5 butthe average concentration of the chromosome 21 locus per well woulddepend on the fractional fetal DNA concentration in the tested sample,as shown in Table 700. The distribution of the reference and/or thechromosome 21 loci to a well was determined by the random numbersrepresenting the respective locus which were generated according to thePoisson probability density function with the appropriate averageconcentration of the locus per well.

In step 2, step 1 was repeated until the desired number of wells hadbeen generated, e.g. 384 wells for a 384-well plate experiment. Thenumbers of wells positive for chromosome 1 only and chromosome 21 onlywere counted. The number of the informative wells was the total numberof wells positive for either one of the chromosomes but not both. Theproportion of informative wells positive for chromosome 21 (P_(r)) wasthen calculated. The upper and lower boundaries of the relevant SPRTcurves to accept the null or alternative hypothesis were calculated asdescribed in the earlier section on SPRT analysis.

In step 3, 5000 simulations were performed for each of the two scenariosof the pregnant woman carrying a euploid or a trisomy 21 fetus. Eachsimulation could be regarded as an independent biological sampleobtained from pregnant women. In Table 1100, the correct classificationof euploid cases refers to those euploid cases in which the nullhypothesis was accepted and the incorrect classification of euploidcases refers to those euploid cases in which the alternative hypothesiswas accepted. Similarly, those trisomy 21 cases in which the alternativehypothesis was accepted were regarded as correctly classified and thosetrisomy 21 cases in which the null hypothesis was accepted were regardedas incorrectly classified. For both groups, those cases in which neitherthe null or alternative hypothesis was accepted after the pre-specifiedtotal number of wells had been simulated were regarded as unclassified.

In step 4, steps 1 to 3 were repeated for samples with 10% 25%, 50% and100% fetal DNA at total well numbers ranging from 384 to 7680.

II. Validation of Trisomy 21 Detection

A. RNA-SNP for PLAC4

The practical feasibility of digital RNA-SNP was demonstrated using thers8130833 SNP on the PLAC4 gene on chromosome 21 (Lo, Y M D et al. 2007Nat Med 13, 218-223). Placental DNA and RNA samples from two euploid andtwo trisomy 21 heterozygous placentas were analyzed. The placental DNAsamples were analyzed with the digital RNA-SNP protocol but with theomission of the reverse transcription step, thus essentially convertingthe procedure to digital DNA-SNP analysis. To strike a balance betweenthe chance of correct case classification and the proportion ofinformative wells, we diluted the samples aiming for one allele of anytype per well and confirmed by a 96-well digital PCR analysis. This wasfollowed by a 384-well digital RNA-SNP experiment. P_(r) and m_(r) werecalculated and the SPRT curve for this m_(r) value was used for datainterpretation.

FIG. 13A shows a table 1300 of digital RNA-SNP analysis in placentaltissues of euploid and trisomy 21 pregnancies according to an embodimentof the present invention. Genotypes were determined by massspectrometric assay. “Euploid” was assigned when the experimentallyobtained P_(r) was below the unclassifiable region; “T21”, representingtrisomy 21, was assigned when the experimentally obtained P_(r) wasabove the unclassifiable region. T21, trisomy 21. Each of these caseswas correctly classified, using both the DNA and RNA samples, with one384-well experiment.

We further tested plasma RNA samples from nine women carrying euploidfetuses and four women carrying trisomy 21 fetuses. FIG. 13B shows atable 1350 of digital RNA-SNP analysis of maternal plasma from euploidand trisomy 21 pregnancies according to an embodiment of the presentinvention. All of the cases were correctly classified. Initial resultsfor one trisomy 21 case (M2272P) fell within the unclassifiable regionbetween the SPRT curves after one 384-well experiment. Thus, anadditional 384-well run was performed. New m_(r) and P_(r) values werecalculated from the aggregated data from the total of 768 wells and theclassification was performed using a new set of SPRT curves selectedbased on this m_(r) value. The case was then scored correctly asaneuploid.

Our experimental and simulation data show that digital RNA-SNP is aneffective and accurate method for trisomy 21 detection. As PLAC4 mRNA inmaternal plasma is derived purely from the fetus, for 12 of the 13maternal plasma samples tested, only one 384-well digital PCR experimentwas required for correct classification. This homogenous, real-timedigital PCR-based approach thus offers an alternative to the massspectrometry-based approach for RNA-SNP analysis. Apart fromplacental-specific mRNA transcripts, we also envision that other typesof fetal-specific nucleic acid species in maternal plasma could be usedfor digital PCR-based detection of fetal chromosomal aneuploidies. Oneexample is fetal epigenetic markers which have recently been used forthe noninvasive prenatal detection of trisomy 18 using the epigeneticallelic ratio (EAR) approach (Tong Y K et al. 2006 Clin Chem, 52,2194-2202). Thus, we predict that digital EAR would be a possibleanalytical technique.

B. RCD

The practical feasibility of digital RCD for trisomy 21 detection wasalso investigated using a PCR assay targeting paralogous sequences onchromosome 21 and 1. Paralogous loci were used here by way of examples.Non-paralogous sequences on chromosome 21 and any other referencechromosome can also be used for RCD. Placental DNA samples from twoeuploid and two trisomy 21 placentas were diluted to approximately onetarget template from either chromosome per well and confirmed by a96-well digital PCR analysis. Each confirmed sample was analyzed by a384-well digital RCD experiment and the values of P_(r) and m_(r) werecalculated. For digital RCD, the chromosome 1 paralog was the referencetemplate. This m_(r) value was used to select a corresponding set ofSPRT curves for data interpretation. All of the placental samples werecorrectly classified as shown in FIG. 14A.

To demonstrate that the digital RCD approach can be used to detecttrisomy 21 DNA which is mixed with an excess of euploid DNA, such as thescenario of fetal DNA in maternal plasma, mixtures containing 50% and25% of trisomy 21 placental DNA in a background of euploid maternalblood cell DNA were analyzed. Placental DNA from 10 trisomy 21 and 10euploid cases was each mixed with an equal amount of euploid maternalblood cell DNA, thus producing twenty 50% DNA mixtures. FIG. 14B shows aplot 1440 illustrating the SPRT interpretation for RCD analysis of the50% fetal DNA mixtures according to an embodiment of the presentinvention. Similarly, placental DNA from 5 trisomy 21 and 5 euploidcases was each mixed with 3 times excess of euploid maternal blood cellDNA, thus producing ten 25% DNA mixtures. FIG. 14C shows a plot 1470illustrating the SPRT interpretation for RCD analysis of the 25% fetalDNA mixtures. All the euploid and aneuploid DNA mixtures were correctlyclassified, as shown in FIGS. 14B and 14C.

Each sample reached the point of being classifiable after a number of384-well digital PCR analyses as marked on FIGS. 14B and 14C. For the50% DNA mixtures, the number of 384-well plates required ranged from oneto five. For the 25% DNA mixtures, the number of 384-well platesrequired ranged form one to seven. The cumulative proportion of casesbeing correctly classified with the addition of every 384 digital PCRanalyses were as predicted by the computer simulation presented in Table1200.

III. Method with Digital PCR

A. Digital RNA-SNP

All RNA samples were first reverse transcribed with a gene-specificreverse transcription primer using the ThermoScript reversetranscriptase (Invitrogen). Sequence of the reverse transcription primerwas 5′-AGTATATAGAACCATGTTTAGGCCAGA-3′ (SEQ ID NO:1) (Integrated DNATechnologies, Coralville, Iowa). The subsequent treatment of the reversetranscribed RNA (i.e. the cDNA) samples for digital RNA-SNP, and DNAsamples (e.g. placental DNA) was essentially the same. Prior to digitalPCR analysis, DNA and the cDNA samples were first quantified using areal-time PCR assay towards PLAC4, consisting of primers5′-CCGCTAGGGTGTCTTTTAAGC-3′ (SEQ ID NO:2),5′-GTGTTGCAATACAAAATGAGTTTCT-3′ (SEQ ID NO:3), and the fluorescent probe5′-(FAM)ATTGGAGCAAATTC(MGBNFQ)-3′ (SEQ ID NO:4) (Applied Biosystems,Foster City, Calif.), where FAM is 6-carboxyfluorescein and MGBNFQ is aminor groove binding non-fluorescent quencher.

A calibration curve was prepared by serial dilutions of HPLC-purifiedsingle-stranded synthetic DNA oligonucleotides (Proligo, Singapore)specifying the amplicon. The sequence was5′-CGCCGCTAGGGTGTCTTTTAAGCTATTGGAGCAAATTCAAATTTGGCTTAAAGAAAAAGAAACTCATTTTGTATTGCAACACCAGGAGTATCCCAAGGGACTCG-3′ (SEQ ID NO:5). Thereaction was set up using 2×TaqMan Universal PCR Master Mix (AppliedBiosystems) in a reaction volume of 25 μL. 400 nM of each primer and 80nM of the probe were used in each reaction. The reaction was initiatedat 50° C. for 2 min, followed by 95° C. for 10 min and 45 cycles of 95°C. for 15 s and 60° C. for 1 min in an ABI PRISM 7900HT SequenceDetection System (Applied Biosystems). Serial dilutions of the DNA orcDNA samples were then undertaken such that the subsequent digital PCRamplification could be performed at approximately one template moleculeper well. At such a concentration, it was expected that approximately37% of the reaction wells would show negative amplification and wasfirst confirmed by conducting a 96-well digital real-time PCR analysis.This was followed by digital RNA-SNP analysis conducted in 384-wellplates using a set of non-intron spanning primers: the forward primer5′-TTTGTATTGCAACACCATTTGG-3′ (SEQ ID NO:6) and the gene-specific reversetranscription primer described above.

Two allele-specific TaqMan probes targeting each of the two alleles ofthe rs8130833 SNP on the PLAC4 sequence were designed. Their sequenceswere 5′-(FAM)TCGTCGTCTAACTTG(MGBNFQ)-3′ (SEQ ID NO:7) and5′-(VIC)ATTCGTCATCTAACTTG(MGBNFQ)-3′ (SEQ ID NO:8) for the G and Aalleles, respectively. The reaction was set up using 2×TaqMan UniversalPCR Master Mix in a reaction volume of 5 μL. Each reaction contains1×TaqMan Universal PCR Master Mix, 572 nM of each primer, 107 nM of theallele-G-specific probe and 357 nM of the allele-A-specific probe. Thereaction was carried out in the ABI PRISM 7900HT Sequence DetectionSystem. The reaction was initiated at 50° C. for 2 min, followed by 95°C. for 10 min and 45 cycles of 95° C. for 15 s and 57° C. for 1 min.During the reaction, the fluorescence data were collected by the“Absolute Quantification” application of the SDS 2.2.2 software (AppliedBiosystems). The software automatically calculated the baselines and thethreshold values. The number of wells which were positive for either theA or the G alleles was recorded and subjected to SPRT analysis.

B. Digital RCD Analysis

All placental and maternal buffy coat DNA samples used in this studywere first quantified by the NanoDrop spectrophotometer (NanoDropTechnology, Wilmington, Del.). The DNA concentration is converted tocopies/4 using a conversion of 6.6 pg/cell. The amount of DNAcorresponding to approximately one template per well was determined byserially diluting the DNA samples and confirmed with the real-time PCRassay in a 96-well format where we expect approximately 37% of the wellsto show negative amplification. The PCR setup for the confirmatory platewas the same as described below except that only the probe for thereference chromosome was added. In the digital RCD analysis, theparalogous loci on chromosome 21 and 1 (Deutsch, S. et al. 2004 J MedGenet 41, 908-915) were first co-amplified by forward primer5′-GTTGTTCTGCAAAAAACCTTCGA-3′ (SEQ ID NO:9) and reverse primer5′-CTTGGCCAGAAATACTTCATTACCATAT-3′ (SEQ ID NO:10). Twochromosome-specific TaqMan probes were designed to target the chromosome21 and 1 paralogs, and their sequences were5′-(FAM)TACCTCCATAATGAGTAAA(MGBNFQ)-3′ (SEQ ID NO:11) and5′-(VIC)CGTACCTCTGTAATGTGTAA(MGBNFQ)-3′ (SEQ ID NO:12), respectively.Each reaction contained 1×TaqMan Universal PCR Master Mix (AppliedBiosystems), 450 nM of each primer, and 125 nM of each probe. The totalreaction volume was 5 μL/well. The reaction was initiated at 50° C. for2 min, followed by 95° C. for 10 min and 50 cycles of 95° C. for 15 sand 60° C. for 1 min. All real-time PCR experiments were carried out onan ABI PRISM 7900HT Sequence Detection System (Applied Biosystems), andthe fluorescence data were collected by the “Absolute Quantification”application of the SDS 2.2.2 software (Applied Biosystems). The defaultbaselines and manual threshold values were used. The number of wellswhich were positive for either chromosome 21 or chromosome 1 wasrecorded and subjected to SPRT analysis. One or more 384-well plateswould be analyzed until disease classification was possible by SPRT.

IV. Using Microfluidics-Based Digital PCR

A. Digital RNA-SNP

This example demonstrates the performance of digital PCR analysis usingmicrofluidics-based digital PCR. A variant of this approach isillustrated here, by way of example but not by way of limitation to,using a Fluidigm BioMark™ System. This system can perform over 9000digital PCRs per run.

Placental tissues and maternal peripheral blood samples were obtainedfrom pregnant women carrying euploid or trisomy 21 fetuses. Genotypingof the rs8130833 SNP on the PLAC4 gene was carried out in placental DNAsamples by primer extension followed by mass spectrometry. RNA wasextracted from the placental and maternal plasma samples.

All RNA samples were reverse transcribed with a gene-specific reversetranscription primer (5′-AGTATATAGAACCATGTTTAGGCCAGA-3; SEQ ID NO:13)using the ThermoScript reverse transcriptase (Invitrogen). For theplacental cDNA samples, serial dilutions were performed such that thesubsequent digital PCR amplification could be performed at approximatelyone template molecule per well.

Digital PCR was conducted on the BioMark System™ (Fluidigm) with a12.765 Digital Array (Fluidigm). Each Digital Array consists of 12panels for accommodating 12 sample-assay mixtures. Each panel is furtherpartitioned into 765 wells for carrying out a 7-nL reaction/well. Thers8130833 SNP region on the PLAC4 gene was amplified by the forwardprimer (5′-TTTGTATTGCAACACCATTTGG-3; SEQ ID NO:14) and the gene-specificreverse transcription primer described above. Two allele-specific TaqManprobes targeting each of the two alleles of the rs8130833 SNP weredesigned. Their sequences were 5′-(FAM)TCGTCGTCTAACTTG(MGBNFQ)-3′ (SEQID NO:15) and 5′-(VIC)ATTCGTCATCTAACTTG(MGBNFQ)-3′ (SEQ ID NO:16) forthe G and A alleles, respectively. The reaction for one array panel wasset up using 2×TaqMan Universal PCR Master Mix in a reaction volume of10 μL. Each reaction contains 1×TaqMan Universal PCR Master Mix, 572 nMof each primer, 53.5 nM of the allele-G-specific probe, 178.5 nM of theallele-A-specific probe and 3.5 μL of the cDNA sample. One reactionpanel was used for each placental cDNA sample while 12 panels were usedfor each maternal plasma sample. The sample-assay mixtures were loadedinto the Digital Array by a NanoFlex™ IFC controller (Fluidigm). Thereaction was carried out in the BioMark™ System. The reaction wasinitiated at 50° C. for 2 min, followed by 95° C. for 10 min and 40cycles of 95° C. for 15 s and 57° C. for 1 min.

Placental RNA samples from one euploid and two T21 heterozygousplacentas were analyzed in a 765-well reaction panel. For each sample,the number of informative wells, comprising the ones positive for eitherthe A or the G allele (but not both), was counted. The proportion of theoverrepresented allele among all the informative wells (P_(r)) wasdetermined. SPRT curves appropriate for the exact average referencetemplate concentration per well (m_(r)) of these runs were applied todetermine if the experimentally-obtained P_(r) indicated a euploid orT21 sample. As shown in FIG. 15A, all RNA samples were correctlyclassified using this approach.

We further tested the plasma RNA samples from four women carryingeuploid fetuses and one woman carrying a trisomy 21 fetus. Each samplewas analyzed in twelve 765-well reaction panels, i.e. 9180 reactions perplasma RNA sample. FIG. 15B shows the number of informative wells foreach of the 12 panels for this plasma RNA sample. As shown in the table,the template concentration in the plasma sample was so diluted that thenumber of informative wells in any one reaction panel was not sufficientfor the SPRT classification. The informative wells from three reactionpanels had to be combined before this sample was classified as a euploidsample (FIG. 15C). FIG. 15C shows that using the aggregated data fromtwo to twelve panels, all of the plasma cases could be correctlyclassified.

Compared with the manual method for performing digital PCR, thismicrofluidics-based method is much more rapid and less labor-intensive.The whole process could be completed in two and a half hours.

B. Digital RNA-SNP Analysis for the Prenatal Detection of Trisomy 18

In this example, we used a digital PCR-based allelic discriminationassay on serpin peptidase inhibitor clade B (ovalbumin) member 2(SERPINB2) mRNA, a placenta-expressed transcript on chromosome 18, todetect an imbalance in the ratio of polymorphic alleles for trisomy 18fetuses. Extraction of DNA and RNA from placental tissue samples wasperformed using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) andthe TRIzol reagent (Invitrogen, Carlsbad, Calif.), respectively, asdescribed in the manufacturers' protocols. The extracted placental RNAsamples were subjected to DNase I treatment (Invitrogen) for removal ofcontaminating genomic DNA. Genotyping of the rs6098 SNP on the SERPINB2gene was carried out in placental tissue DNA samples with a HomogenousMassEXTEND (hME) assay using the MassARRAY Compact (Sequenom, San Diego)as previously described.

Reverse transcription for the SERPINB2 transcript was performed on theplacental tissue RNA samples with a gene-specific primer5′-CGCAGACTTCTCACCAAACA-3′ (SEQ ID NO:17) (Integrated DNA Technologies,Coralville, Iowa) using the ThermoScript reverse transcriptase(Invitrogen). All cDNA samples were diluted to a concentration such thatthe subsequent digital PCR amplification could be performed at anaverage concentration of one template molecule per reaction well.Digital PCR was set up using the TaqMan Universal PCR Master Mix(Applied Biosystems, Foster City, Calif.) and the Biomark™ PCR Reagents(Fluidigm, San Francisco). The forward primer 5′-CTCAGCTCTGCAATCAATGC-3′(SEQ ID NO:18) (Integrated DNA Technologies) and the reverse primer(identical to the gene-specific primer used for reverse transcription)were used at a concentration of 600 nM. The two TaqMan probes targetingthe A or G allele of the rs6098 SNP on the SERPINB2 sequence were5′-(FAM)CCACAGGGAATTATTT (MGBNFQ)-3′ (SEQ ID NO:19) and5′-(VIC)CCACAGGGGATTATTT(MGBNFQ)-3′ (SEQ ID NO:20) (Applied Biosystems).FAM is 6-carboxyfluorescein and MGBNFQ is a minor groove-bindingnonfluorescent quencher, and were used at concentrations of 300 nM and500 nM, respectively. Each sample-reagent mix was partitioned into 765reaction wells on a Biomark™ 12.765 Digital Array using the Nanoflex™IFC Controller (Fluidigm). After partitioning, the array was placed inthe Biomark™ Real-time PCR System (Fluidigm) for thermal amplificationand fluorescence detection. The reaction was initiated at 50° C. for 2min and continued at 95° C. for 5 min followed by 45 cycles of 95° C.for 15 sec and 59° C. for 1 min. After amplification, the number ofinformative wells (one that was positive for either the A or G alleleonly) and the number of wells positive for both alleles were counted andsubjected to sequential probability ratio test (SPRT) analysis.

For a heterozygous euploid fetus, the A and G alleles should be equallyrepresented (1:1) in the fetal genome, whereas for trisomy 18, therewould be an additional copy of one allele thus giving a ratio of 2:1 inthe fetal genome. A series of SPRT curves were generated forinterpretation of different samples. These curves illustrate theexpected proportion of informative wells positive for theoverrepresented allele P_(r) (y-axis) for a given total number ofinformative wells (x-axis) needed for classification. For each sample,the experimentally derived P_(r) was compared with the expected P_(r)value. Samples above the upper curve were classified as trisomy 18,whereas those below the bottom curve were classified as euploid. Thearea between the two curves is the unclassifiable region.

The feasibility of digital RNA-SNP analysis for the detection of fetaltrisomy 18 was demonstrated by using the rs6098 SNP on the SERPINB2gene. Placental tissue DNA samples from subjects with euploid andtrisomy 18 fetuses were first genotyped by mass spectrometry foridentifying heterozygous cases. Nine euploid and three trisomy 18heterozygous placentas were found and subjected to digital RNA-SNPanalysis. For each sample, P_(r) and m_(r) were calculated, and the SPRTcurve for this m_(r) value was used for disease classification. As shownin FIG. 16A, all samples were correctly classified. The P_(r) values fortrisomy 18 placentas were above the unclassifiable region, whereas thosefor euploid placentas fell below this region.

Samples with SPRT curves based on m_(r)=0.1, 0.2, and 0.3 areillustrated in FIG. 16B. These data suggest that the digital RNA-SNPmethod is a valuable diagnostic tool for trisomy 18 pregnancies. The twocurves represent the boundaries for the unclassifiable region. Sampleswith data points above the upper curve were classified as aneuploid,whereas those with data points below the bottom curve were classified aseuploid.

C. Digital RCD Analysis

This example demonstrates the performance of digital RCD analysis usingmicrofluidics-based digital PCR. A variant of this approach isillustrated here, by way of example but not by way of limitation to,using a Fluidigm BioMark™ System. This system can perform over 9000digital PCRs per run.

Placental tissues, maternal blood cell and amniotic fluid samples wereobtained from pregnant women carrying euploid or trisomy 21 (T21)fetuses. Placental DNA from 10 T21 and 10 euploid cases was each mixedwith an equal amount of euploid maternal blood cell DNA, thus producingtwenty 50% DNA mixtures. To ensure accurate fetal proportion in themixture samples, the extracted DNA was first quantified by opticaldensity (OD) measurement at 260 nm. They were then digitally quantifiedby the BioMark™ System (Fluidigm) using the 12.765 Digital Arrays(Fluidigm). The assay for quantifying the samples was the same asdescribed below except that only the probe for the reference chromosomewas used.

The chromosome dosage in the 50% DNA mixtures and amniotic fluid sampleswas determined by digital PCR analysis of a nonpolymorphic chromosome 21locus relative to one located on chromosome 1. A 101-bp amplicon of apair of paralogous loci on chromosome 21 and 1 was first co-amplified byforward primer 5′-GTTGTTCTGCAAAAAACCTTCGA-3′ (SEQ ID NO:21) and reverseprimer 5′-CTTGGCCAGAAATACTTCATTACCATAT-3′ (SEQ ID NO:22). Twochromosome-specific TaqMan probes were designed to distinguish betweenthe chromosome 21 and 1 paralogs, and their sequences were5′-(FAM)TACCTCCATAATGAGTAAA(MGBNFQ)-3′ (SEQ ID NO:23) and5′-(VIC)CGTACCTCTGTAATGTGTAA(MGBNFQ)-3′ (SEQ ID NO:24), respectively.The use of paralogous loci was used here by way of example only. Inother words, non-paralogous loci could also be used for such analysis.

In order to demonstrate the use of the digital RCD approach to detecttrisomy 18 (T18), another assay targeting paralogous sequences onchromosome 21 and 18 was designed. A 128-bp amplicon of the paralogousloci on chromosome 21 and 18 was first co-amplified by forward primer5′-GTACAGAAACCACAAACTGATCGG-3′ (SEQ ID NO:25) and reverse primer5′-GTCCAGGCTGTGGGCCT-3′ (SEQ ID NO:26). Two chromosome-specific TaqManprobes were designed to distinguish between the chromosome 21 and 18paralogs, and their sequences were 5′-(FAM)AAGAGGCGAGGCAA(MGBNFQ)-3′(SEQ ID NO:27) and 5′-(VIC)AAGAGGACAGGCAAC(MGBNFQ)-3′ (SEQ ID NO:28),respectively. The use of paralogous loci was used here by way of exampleonly. In other words, non-paralogous loci could also be used for suchanalysis.

All experiments were carried out on the BioMark™ System (Fluidigm) usingthe 12.765 Digital Arrays (Fluidigm). The reaction for one panel was setup using 2×TaqMan Universal PCR Master Mix (Applied Biosystems) in areaction volume of 10 μL. Each reaction contained 1×TaqMan Universal PCRMaster Mix, 900 nM of each primer, 125 nM of each probe and 3.5 μL of a50% placental/maternal blood cell DNA sample. The sample/assay mixturewas loaded into the Digital Array by the NanoFlex™ IFC controller(Fluidigm). The reaction was carried out on the BioMark™ System fordetection. The reaction was initiated at 50° C. for 2 min, followed by95° C. for 10 min and 40 cycles of 95° C. for 15 s and 57° C. for 1 min.

The euploid and T21 50% placental/maternal blood cell DNA samples wereanalyzed on the digital arrays with the chr21/chr1 assay. For eachsample, the number of informative wells, comprising the ones positivefor only chr21 or chr1 markers (but not both), was counted. Theproportion of the overrepresented marker among all the informative wells(P_(r)) was determined. SPRT curves appropriate for the exact averagereference template concentration per well (m_(r)) for any one of thedigital PCR panels were applied to determine if theexperimentally-obtained P_(r) indicated a euploid or T21 sample. Datawere aggregated from extra panels for samples which remainedunclassified until a decision could be made. As shown in FIG. 17, all50% placental/maternal blood cell DNA samples were correctly classifiedusing this approach with data ranging from one to four panels needed. ASPRT curve was also plotted to show the decision boundaries for correctclassification, as shown in FIG. 18.

We further applied the RCD analysis on amniotic fluid samples obtainedfrom 23 women carrying euploid fetuses and 6 women carrying T21 fetuses.Each sample was analyzed in a single 765-well reaction panel with thechr21/chr1 assay. FIG. 19 shows the SPRT classification summary. Asshown in FIG. 19, all the 29 samples were classified correctly. Thedigital RCD method is thus an alternative approach for the detection oftrisomies using microsatellite (Levett L J, et al. A large-scaleevaluation of amnio-PCR for the rapid prenatal diagnosis of fetaltrisomy. Ultrasound Obstet Gynecol 2001; 17: 115-8) or single nucleotidepolymorphism (SNP) (Tsui N B, et al. Detection of trisomy 21 byquantitative mass spectrometric analysis of single-nucleotidepolymorphisms. Clin Chem 2005; 51: 2358-62) markers or real-timenon-digital PCR (Zimmermann B, et al. Novel real-time quantitative PCRtest for trisomy 21. Clin Chem 2002; 48: 362-3) in miscellaneous sampletypes used for prenatal diagnosis, such as amniotic fluid and chorionicvillus biopsies.

In an attempt to detect T18 cases, we applied the chr21/chr18 assay on 3euploid and 5 T18 placental DNA samples. The proportion of theoverrepresented marker among all the informative wells (P_(r)) wascalculated. All of them were classified correctly except one T18 casewas misclassified as euploid. The results were summarized in FIG. 20.

V. Using Multiplex Digital RCD Assays on Mass-Spectrometric Platform

The number of plasma DNA molecules that are present per unit volume ofmaternal plasma is limited (Lo Y M D. et al. 1998 Am J Hum Genet 62,768-7758). For example, in early pregnancy, the median maternal plasmaconcentration of an autosomal locus, the β-globin gene, has been shownto be 986 copies/mL, with contributions from both the fetus and mother(Lo Y M D. et al. 1998 Am J Hum Genet 62, 768-7758). To capture 7,680molecules, DNA extracted from some 8 mL of maternal plasma would beneeded. This volume of plasma, obtainable from ˜15 mL of maternal blood,is at the limit of routine practice. However, we envision that multiplesets of chr21 and reference chromosome targets can be combined fordigital RCD analysis. For 5 pairs of chr21 and reference chromosometargets, just 1.6 mL of maternal plasma would be needed to provide thenumber of template molecules needed for analysis. Multiplex singlemolecule PCR could be performed. The robustness of such multiplex singlemolecule analysis has been demonstrated previously for single moleculehaplotyping (Ding, C. and Cantor, C R. 2003 Proc Natl Acad Sci USA 100,7449-7453).

In one example, placental tissues and maternal blood cell samples wereobtained from pregnant women carrying euploid or trisomy 21 (T21)fetuses. 5 euploid and 5 T21 placental DNA samples were each mixed withequal proportions of maternal blood cell DNA to produce 10 DNA mixturesmimicking plasma samples with 50% fetal DNA. To ensure accurate fetalproportion in the mixture samples, the extracted DNA was firstquantified by optical density (OD) measurement at 260 nm. They were thendigitally quantified by real-time PCR in 384-well format. The assay forquantifying the samples was the same as described in the previousexample of digital RCD analysis.

The chromosome dosage in the 50% mix was determined by digital PCRanalysis of a nonpolymorphic chromosome 21 locus relative to one locatedon chromosome 1. The method is called Digital Relative Chromosome Dosage(RCD) analysis. A 121-bp amplicon (inclusive of a 10-mer on each primer)of a pair of paralogous loci on chromosome 21 and 1 was co-amplified byforward primer 5′-ACGTTGGATGGTTGTTCTGCAAAAAACCTTCGA-3′ (SEQ ID NO:29)and reverse primer 5′-ACGTTGGATGCTTGGCCAGAAATACTTCATTACCATAT-3′ (SEQ IDNO:30). An extension primer which targets the base differences betweenchromosome 21 and chromosome 1 was designed, and its sequence is5′-CTCATCCTCACTTCGTACCTC-3′ (SEQ ID NO:31).

In order to demonstrate the utility of multiplexing digital PCR assaysto detect T21 cases, another digital RCD assay targeting paralogoussequences on chromosome 21 and 18 was designed. A 148-bp amplicon(inclusive of a 10-mer on each primer) of the paralogous loci onchromosome 21 and 18 was co-amplified by forward primer5′-ACGTTGGATGGTACAGAAACCACAAACTGATCGG-3′ (SEQ ID NO:32) and reverseprimer 5′-ACGTTGGATGGTCCAGGCTGTGGGCCT-3′ (SEQ ID NO:33). An extensionprimer which targets the base differences between chromosome 21 andchromosome 18 was designed, and its sequence is 5′-ACAAAAGGGGGAAGAGG-3′(SEQ ID NO:34).

Multiplex digital RCD analysis was performed using primer extensionprotocol. PCR reaction was set up using GeneAmp PCR Core Reagent Kit(Applied Biosystems) in a reaction volume of 5 μL. Each reactioncontained 1×Buffer II, 2 mM MgCl₂, 200 μM dNTP mix, 0.2 U AmpliTaq Gold,200 nM of each of the 4 primers and the 50% DNA mix. The assay/samplemixture was dispensed into 384-well PCR plate and the reaction wasinitiated at 50° C. for 2 min, followed by 95° C. for 10 min and 40cycles of 95° C. for 15 s and 57° C. for 1 min.

PCR products were subjected to shrimp alkaline phosphatase (SAP)treatment to remove unincorporated dNTPs. The mixture was incubated at37° C. for 40 min followed by 85° C. for 5 min. Primer extensionreaction was then carried out. In brief, 771 nM of extension primer fromchr21/chr1 assay, 1.54 μM of extension primer from chr21/chr18 assay,0.67 U Thermosequenase (Sequenom), and 64 μM each of ddCTP, ddGTP, dATPand dTTP in an extension cocktail were added to the SAP-treated PCRproducts. The reaction conditions were 94° C. for 2 min, followed by 94°C. for 5 s, 50° C. for 5 s, and 72° C. for 5 s for 80 cycles. 16 μL ofwater and 3 mg of the Clean Resin (Sequenom) were added to the extensionproducts for a final clean up. The mixtures were mixed in a rotator for20 to 30 min, followed by a centrifugation step at 361 g for 5 min.Fifteen to 25 nL of the final products were dispensed onto a SpectroCHIP(Sequenom) by a MassARRAY Nanodispenser S (Sequenom). Data acquisitionfrom the SpectroCHIP was done in the MassARRAY Analyzer Compact MassSpectrometer (Sequenom). Mass data were imported into the MassARRAYTyper (Sequenom) software for analysis.

The five euploid and five T21 50% placental/maternal DNA samples wereanalyzed with the duplex RCD assay. For each sample, the number ofinformative wells from individual assay, comprising the ones positivefor only chr21 or chr1 or chr18 markers, was counted. The proportion ofthe chr21 marker among all the informative wells (P_(r)) was calculatedseparately for each RCD assay. Sequential probability ratio test (SPRT)was then applied to determine if the P_(r) indicated a euploid or T21sample. By doing so, the number of wells required was reduced as eachplate was counted twice.

The chr21/chr1 assay was usually applied first. If the sample remainedunclassified, then the values from the chr21/chr18 assay would be addedfor further calculations. Extra plates were used for samples whichremained unclassified until a decision could be made. As shown in FIGS.21A and 21B, all euploid 50% mix samples were correctly classified usinga single 384-well plate. Several T21 cases required 2 or more plates forcorrect classification. If only one assay was used, a greater number ofplates would be needed to attain the number of informative wellsrequired when classification was achieved. For example, data for caseN0230 was unclassifiable when either of the RCD assays was used alone.However, correct classification was achieved when data from the twoassays were combined. If the duplex RCD assays were not used, additionalplates of analyses would be needed. We would expect a further reductionof well number with a higher level of multiplexing of assays.

In another example, we developed a 4-plex assay targeting 4 differentamplicons on chromosome 21 and their corresponding paralogous partnerslocated on other non-chromosome-21 autosomes. This 4-plex assay was usedin digital RCD analysis followed by SPRT classification of samples fromeuploid and trisomy 21 pregnancies. DNA extractions from placentalsamples were performed using the QIAamp tissue kit (Qiagen, Hilden,Germany).

All placental and maternal buffy coat DNA samples used in this studywere first quantified by the NanoDrop spectrophotometer (NanoDropTechnology, Wilmington, Del.). The DNA concentration was converted togenome equivalent (GE)/4 using a conversion of 6.6 pg/cell. The amountof DNA corresponding to approximately one template per well wasdetermined by serially diluting the DNA samples. Under such a condition,we would expect approximately 37% of the wells to show negativeamplification. In multiplex digital RCD analysis, 4 sets of paralogoussequence targets were selected: the paralogous loci on chromosome 21 and1 were co-amplified by the forward primer5′-ACGTTGGATGTTGATGAAGTCTCATCTCTACTTCG-3′ (SEQ ID NO:35) and the reverseprimer 5′-ACGTTGGATGCAATAAGCTTGGCCAGAAATACT-3′ (SEQ ID NO:36), resultingin an amplicon of 81 bp. The paralogous loci on chromosome 21 and 7 wereco-amplified by the forward primer5′-ACGTTGGATGGAATTTAAGCTAAATCAGCCTGAACTG-3′ (SEQ ID NO:37) and thereverse primer 5′-ACGTTGGATGGTTTCTCATAGTTCATCGTAGGCTTAT-3′ (SEQ IDNO:38), resulting in an amplicon of 82 bp. The paralogous loci onchromosome 21 and 2 were co-amplified by the forward primer5′-ACGTTGGATGTCAGGCAGGGTTCTATGCAG-3′ (SEQ ID NO:39) and the reverseprimer 5′-ACGTTGGATGAGGCGGCTTCCTGGCTCTTG-3′ (SEQ ID NO:40), resulting inan amplicon of 101 bp. The paralogous loci on chromosome 21 and 6 wereco-amplified by the forward primer 5′-ACGTTGGATGGCTCGTCTCAGGCTCGTAGTT-3′(SEQ ID NO:41) and the reverse primer5′-ACGTTGGATGTTTCTTCGAGCCCTTCTTGG-3′ (SEQ ID NO:42), resulting in anamplicon of 102 bp. Each reaction contained 10×buffer II (AppliedBiosystems), MgCl₂ and 100 nM of each primer. The total reaction volumewas 5 μL/well. The reaction was initiated at 95° C. for 5 min, followedby 45 cycles of 95° C. for 30 sec, 62° C. for 30 sec and 72° C. for 30sec, and a final extension at 72° C. for 7 min. All conventional PCRamplifications were carried out on a GeneAmp PCR System 9700 (AppliedBiosystems). The unincorporated nucleotides were deactivated by shrimpalkaline phosphatase (SAP) treatment. Each reaction contained 10×SAPbuffer (Sequenom) and SAP enzyme (Sequenom). 2 μL of SAP mix was addedto each PCR. The SAP reaction was incubated at 37° C. for 40 min and 85°C. for 5 min. After the SAP treatment, primer extension reaction wascarried out on the PCR products using the iPLEX Gold kit (Sequenom). Theparalogous sequence mismatches (PSMs) on the paralogous loci onchromosomes 21 and 1 were interrogated by the extension primer5′-GTCTCATCTCTACTTCGTACCTC-3′ (SEQ ID NO:43). The PSMs on the paralogousloci on chromosomes 21 and 7 were interrogated by the extension primer5′-TTTTACGCTGTCCCCATTT-3′ (SEQ ID NO:44). The PSMs on the paralogousloci on chromosomes 21 and 2 were interrogated by the extension primer5′-GGTCTATGCAGGAGCCGAC-3′ (SEQ ID NO:45). The PSMs on the paralogousloci on chromosomes 21 and 6 were interrogated by the extension primer5′-TGGGCGCGGGAGCGGACTTCGCTGG-3′ (SEQ ID NO:46). Each reaction contained10×iPLEX buffer (Sequenom), iPLEX termination mix (Sequenom), iPLEXenzyme (Sequenom) and 343 nM of each extension primer, except for theextension primer for PSMs on chromosomes 21 and 6 which was used at 1.03μM. 2 μL of iPLEX mix was added to 5 μL of PCR product. The iPLEXreaction was cycled according to a 200-short-cycle program. Briefly, thesamples were first denatured at 94° C. for 35 sec, followed by annealingat 52° C. for 5 sec and extension at 80° C. for 5 sec. The annealing andextension cycle was repeated four more times for a total of five cyclesand then looped back to a 94° C. denaturing step for 5 sec, after whichwas the 5-cycle annealing and extension loop again. The five annealingand extension cycles with the single denaturing step were repeated 39times for a total of 40. A final extension at 72° C. for 3 min wasperformed. The iPLEX reaction products were diluted with 16 μL water anddesalted by 6 mg resin for each PCR. The 384-well plate was centrifugedat 1600 g for 3 min before dispensing onto the SpectroCHIP (Sequenom)and the matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry MS Analysis (Sequenom).

The number of wells which were positive for only chromosome 21 or onlythe reference chromosome for each of the four assays was independentlyrecorded. For each assay, the Poisson corrected numbers of molecules forchromosome 21 and the reference chromosome were calculated. The sum ofthe Poisson corrected number of molecules for chromosome 21 as well asthe sum of the Poisson corrected number of reference chromosomes fromall four assays were calculated and deemed as the informative counts forthe 4-plex assay. The P_(r) value was the chromosome 21 count for the4-plex assay divided by the sum of the chromosome 21 and referencechromosome counts for the 4-plex assay. The experimentally derived P_(r)values were subjected to SPRT analysis. One or more 384-well plateswould be analyzed until disease classification was possible by SPRT. Atotal of two 50% euploid placental genomic DNA/50% maternal buffy coatDNA mix and two 50% trisomy 21 placental genomic DNA/50% maternal buffycoat DNA mix were analyzed.

The experimentally derived P_(r) value would be compared with theexpected value of P_(r) to test the null or alternative hypotheses.Alternatively, neither the null or alternative hypothesis could beaccepted if the P_(r) for the given number of informative counts has notyet reached the required level of statistical confidence for diseaseclassification. These cases were deemed unclassifiable until more datawere available.

The results and the SPRT classification of each sample are tabulated inFIGS. 22A and 22B. The two euploid samples required 2 and 5 384-wellmultiplex digital RCD analyses before SPRT classification could bereached. Data from none of the individual member of the 4-plex assayallowed disease classification by SPRT. Both trisomy 21 samples wereeach correctly classified with just one 384-well multiplex digital RCDanalysis. Similarly, data from none of the individual member of the4-plex assay allowed disease classification by SPRT. However, thecomposite counts from the 4-plex assay allowed correct SPRTclassification. These data illustrated that by using multiplex digitalRCD, the effective number of informative counts were substantiallyincreased for a given number of digital PCR analyses performed ascompared to the use of a single-plex digital RCD assay.

VI. Using Digital Epigenetic Relative Chromosome Dosage

Here we outline an approach called digital epigenetic relativechromosome dosage (digital ERCD) in which epigenetic markers exhibitinga fetal-specific DNA methylation pattern, or other epigenetic changes,on a chromosome involved in a chromosomal aneuploidy (e.g. chromosome 21in trisomy 21) and on a reference chromosome, are subjected to digitalPCR analysis. The ratio of the number of wells positive for thechromosome 21 epigenetic marker to that positive for the referencechromosome epigenetic marker in plasma DNA extracted from pregnant womenbearing normal fetuses will give us the reference range. The ratio willbe expected to be increased if the fetus has trisomy 21. It is obviousto those of skill in the art that more than one chromosome 21 markersand more than one reference chromosome markers could be used in thisanalysis.

One example of a gene on chromosome 21 which exhibits a fetus(placenta)-specific methylation pattern is the Holocarboxylasesynthetase (HLCS) gene. HLCS is hypermethylated in the placenta, buthypomethylated in maternal blood cells; and is covered in U.S. patentapplication Ser. No. 11/784,499, which is incorporated herein byreference. One example of a gene on a reference chromosome whichexhibits a fetus (placenta)-specific methylation pattern is the RASSF1Agene on chromosome 3 [10]. RASSF1A is hypermethylated in the placentabut is hypomethylated in maternal blood cells, see U.S. patentapplication Ser. No. 11/784,501, which is incorporated herein byreference.

In the application of hypermethylated HLCS and hypermethylated RASSF1Ato digital PCR detection of trisomy 21 in a fetus using maternal plasma,maternal peripheral blood is first collected. Then the blood issubjected to centrifugation and the plasma is harvested. DNA from theplasma is then extracted using techniques well-known to those of skillin the art, such as using a QIAamp Blood kit (Qiagen). The plasma DNA isthen subjected to digestion using one or more methylation-sensitiverestriction enzymes, such as HpaII and BstUI. Thesemethylation-sensitive restriction enzyme(s) will cut the maternal,nonmethylated versions of these genes, while leaving the fetalhypermethylated sequences intact. The digested plasma DNA sample is thendiluted to an extent that on average approximately 0.2 to 1 molecule ofeither the restriction enzyme treated but intact HLCS or RASSF1Asequences will be detected per reaction well. Two real-time PCR systemswill be used to amplify the diluted DNA, one with two primers and oneTaqMan probe specific to the HLCS gene, encompassing the region thatwill be cut by the restriction enzyme(s) if the sequence inunmethylated; and the other one towards the RASSF1A gene, similarly withtwo primers and one TaqMan probe. With regard to the latter RASSF1Aprimer/probe set, one example has been described by Chan et al 2006,Clin Chem 52, 2211-2218. The TaqMan probes towards the HLCS and RASSF1Atargets will have different fluorescent reporters, such as FAM and VIC,respectively. A 384-well plate is then used to perform the digital PCRexperiment. The number of wells scored positive for just HLCS and thosescored positive for just RASSF1A will be counted, and a ratio of thesecounts will be taken. The HLCS:RASSF1A ratio will be expected to behigher for maternal plasma taken from a pregnant woman carrying atrisomy 21 fetus, when compared with one carrying a normal euploidfetus. The degree of overrepresentation will be dependent on the averagereference template concentration per well in the digital PCR run.

Other methods for scoring these results will be possible, for theexample the counting of the number of wells positive for HLCS,irrespective of the concurrent positivity for RASSF1A; and vice versafor RASSF1A, irrespective of the concurrent positivity for HLCS.Furthermore, in replacement of calculating the ratio, either the totalnumber, or the difference in the HLCS and RASSF1A counts could be usedto indicate the trisomy 21 status of a fetus.

Apart from doing the digital PCR in plates, it will also be obvious tothose of skill in the art that other variants of digital PCR can beused, e.g. microfluidics chips, nanoliter PCR microplate systems,emulsion PCR, polony PCR and rolling-circle amplification, primerextension and mass spectrometry, etc. These variants of digital PCR arenamed by way of examples, and not as limitations.

Apart from real-time PCR, it will also be obvious to those of skill inthe art that methods such as mass spectrometry can be used to score thedigital PCR results.

Apart from using methylation-sensitive restriction enzymes todifferentiate the fetal and maternal versions of HLCS and RASSF1A, itwill be obvious to those of skill in the art that other methods forascertaining the methylation status would also be applicable, e.g.bisulfite modification, methylation-specific PCR, immunoprecipitationusing antibody to methylated cytosine, mass spectrometry, etc.

It will also be obvious to those of skill in the art that the approachillustrated in this example and other examples in this patentapplication can be used in the other bodily fluids in which fetal DNAmay be found, including maternal urine, amniotic fluid, transcervicalwashings, chorionic villus, maternal saliva, etc.

VII. Massively Parallel Genomic Sequencing Using Emulsion PCR and OtherStrategies

Here we shall describe another example whereby a digital readout ofnucleic acid molecules can be used for the detection of fetalchromosomal aneuploidies, e.g. trisomy 21, in maternal plasma. Fetalchromosomal aneuploidy results from abnormal dose(s) of a chromosome orchromosomal region. It is desirable that noninvasive tests have highsensitivity and specificity to minimize false diagnoses. However, fetalDNA is present in low absolute concentration and represent a minorportion of all DNA sequences in maternal plasma and serum. Hence, thenumber of digital PCR sampling targeting specific gene loci cannot beincreased infinitely within the same biological specimen. Hence, theanalysis of multiple sets of specific target loci may be used toincrease the amount of data that could be obtained from a specimenwithout increasing the number of digital PCR sampling performed.

Accordingly, embodiments allow the noninvasive detection of fetalchromosomal aneuploidy by maximizing the amount of genetic informationthat could be inferred from the limited amount of fetal nucleic acidswhich exist as a minor population in a biological sample containingmaternal background nucleic acids. In one aspect, the amount of geneticinformation obtained is sufficient for accurate diagnosis yet not overlyexcessive so as to contain costs and the amount of input biologicalsample required.

Massively parallel sequencing, such as that achievable on the 454platform (Roche) (Margulies, M. et al. 2005 Nature 437, 376-380),Illumina Genome Analyzer (or Solexa platform) or SOLiD System (AppliedBiosystems) or the Helicos True Single Molecule DNA sequencingtechnology (Harris T D et al. 2008 Science, 320, 106-109), the singlemolecule, real-time (SMRT™) technology of Pacific Biosciences, andnanopore sequencing (Soni G V and Meller A. 2007 Clin Chem 53:1996-2001), allow the sequencing of many nucleic acid molecules isolatedfrom a specimen at high orders of multiplexing in a parallel fashion(Dear Brief Funct Genomic Proteomic 2003; 1: 397-416). Each of theseplatforms sequences clonally expanded or even non-amplified singlemolecules of nucleic acid fragments.

As a high number of sequencing reads, in the order of hundred thousandsto millions or even possibly hundreds of millions or billions, aregenerated from each sample in each run, the resultant sequenced readsform a representative profile of the mix of nucleic acid species in theoriginal specimen. For example, the haplotype, trascriptome andmethylation profiles of the sequenced reads resemble those of theoriginal specimen (Brenner et al Nat Biotech 2000; 18: 630-634; Tayloret al Cancer Res 2007; 67: 8511-8518). Due to the large sampling ofsequences from each specimen, the number of identical sequences, such asthat generated from the sequencing of a nucleic acid pool at severalfolds of coverage or high redundancy, is also a good quantitativerepresentation of the count of a particular nucleic acid species orlocus in the original sample.

In one embodiment, random sequencing is performed on DNA fragments thatare present in the plasma of a pregnant woman, and one obtains genomicsequences which would originally have come from either the fetus or themother. Random sequencing involves sampling (sequencing) a randomportion of the nucleic acid molecules present in the biological sample.As the sequencing is random, a different subset (fraction) of thenucleic acid molecules (and thus the genome) may be sequenced in eachanalysis. Embodiments will work even when this subset varies from sampleto sample and from analysis to analysis, which may occur even using thesame sample. Examples of the fraction are about 0.1%, 0.5%, or 1% of thegenome. In other embodiments, the fraction is at least any one of thesevalues.

A bioinformatics procedure may then be used to locate each of these DNAsequences to the human genome. It is possible that a proportion of suchsequences will be discarded from subsequent analysis because they arepresent in the repeat regions of the human genome, or in regionssubjected to inter-individual variations, e.g. copy number variations.An amount of the chromosome of interest and of one or more otherchromosomes may thus be determined.

In one embodiment, a parameter (e.g. a fractional representation) of achromosome potentially involved in a chromosomal aneuploidy, e.g.chromosome 21 or chromosome 18 or chromosome 13, may then be calculatedfrom the results of the bioinformatics procedure. The fractionalrepresentation may be obtained based on an amount of all of thesequences (e.g. some measure of all of the chromosomes) or a particularsubset of chromosomes (e.g. just one other chromosome than the one beingtested.)

This fractional representation is then compared to a reference rangeestablished in pregnancies involving normal (i.e. euploid) fetuses. Itis possible that in some variants of the procedure, the reference rangewould be adjusted in accordance with the fractional concentration offetal DNA (f) in a particular maternal plasma sample. The value off canbe determined from the sequencing dataset, e.g. using sequences mappableto the Y chromosome if the fetus is male. The value off may also bedetermined in a separate analysis, e.g. using fetal epigenetic markers(Chan K C A et al 2006 Clin Chem 52, 2211-8) or from the analysis ofsingle nucleotide polymorphisms.

In one aspect, even when a pool of nucleic acids in a specimen issequenced at <100% genomic coverage, and among the proportion ofcaptured nucleic acid molecules, most of each nucleic acid species isonly sequenced once, dosage imbalance of a particular gene locus orchromosome can also be quantitatively determined. In other words, thedosage imbalance of the gene locus or chromosome is inferred from thepercentage representation of the said locus among all mappable sequencedtags of the specimen.

In one aspect for the massively parallel genomic sequencing approach,representative data from all of the chromosomes may be generated at thesame time. The origin of a particular fragment is not selected ahead oftime. The sequencing is done at random and then a database search may beperformed to see where a particular fragment is coming from. This iscontrasted from situations when a specific fragment from chromosome 21and another one from chromosome 1 are amplified.

In one example, a proportion of such sequences would be from thechromosome involved in an aneuploidy such as chromosome 21 in thisillustrative example. Yet other sequences from such a sequencingexercise would be derived from the other chromosomes. By taking intoaccount of the relative size of chromosome 21 compared with the otherchromosomes, one could obtain a normalized frequency, within a referencerange, of chromosome 21-specific sequences from such a sequencingexercise. If the fetus has trisomy 21, then the normalized frequency ofchromosome 21-derived sequences from such a sequencing exercise willincrease, thus allowing the detection of trisomy 21. The degree ofchange in the normalized frequency will be dependent on the fractionalconcentration of fetal nucleic acids in the analyzed sample.

In one embodiment, we used the Illumina Genome Analyzer for single-endsequencing of human genomic DNA and human plasma DNA samples. TheIllumina Genome Analyzer sequences clonally-expanded single DNAmolecules captured on a solid surface termed a flow cell. Each flow cellhas 8 lanes for the sequencing of 8 individual specimens or pools ofspecimens. Each lane is capable of generating ˜200 Mb of sequence whichis only a fraction of the 3 billion basepairs of sequences in the humangenome. Each genomic DNA or plasma DNA sample was sequenced using onelane of a flow cell. The short sequence tags generated were aligned tothe human reference genome and the chromosomal origin was noted. Thetotal number of individual sequenced tags aligned to each chromosomewere tabulated and compared with the relative size of each chromosome asexpected from the reference human genome or non-disease representativespecimens. Chromosome gains or losses were then identified.

The described approach is only one exemplification of the presentlydescribed gene/chromosome dosage strategy. Alternatively, paired endsequencing could be performed. Instead of comparing the length of thesequenced fragments from that expected in the reference genome asdescribed by Campbell et al (Nat Genet 2008; 40: 722-729), the number ofaligned sequenced tags were counted and sorted according to chromosomallocation. Gains or losses of chromosomal regions or whole chromosomeswere determined by comparing the tag counts with the expected chromosomesize in the reference genome or that of a non-disease representativespecimen

In another embodiment, the fraction of the nucleic acid pool that issequenced in a run is further sub-selected prior to sequencing. Forexample, hybridization based techniques such as oligonucleotide arraycould be used to first sub-select for nucleic acid sequences fromcertain chromosomes, e.g. a potentially aneuploid chromosome and otherchromosome(s) not involved in the aneuploidy tested. Another example isthat a certain sub-population of nucleic acid sequences from the samplepool is sub-selected or enriched prior to sequencing. For example, ithas been reported that fetal DNA molecules in maternal plasma arecomprised of shorter fragments than the maternal background DNAmolecules (Chan et al Clin Chem 2004; 50: 88-92). Thus, one may use oneor more methods known to those of skill in the art to fractionate thenucleic acid sequences in the sample according to molecule size, e.g. bygel electrophoresis or size exclusion columns or by microfluidics-basedapproach. Yet, alternatively, in the example of analyzing cell-freefetal DNA in maternal plasma, the fetal nucleic acid portion could beenriched by a method that suppresses the maternal background, such as bythe addition of formaldehyde (Dhallan et al JAMA 2004; 291: 1114-9).

Other single molecule sequencing strategies such as that by the Roche454 platform, the Applied Biosystems SOLiD platform, the the HelicosTrue Single Molecule DNA sequencing technology, the single molecule,real-time (SMRT™) technology of Pacific Biosciences, and nanoporesequencing could similarly be used in this application.

Examples of results and a further discussion (e.g. for sequencing andcalculating parameters) may be found in concurrently filed application“DIAGNOSING FETAL CHROMOSOMAL ANEUPLOIDY USING GENOMIC SEQUENCING,”(Attorney Docket No. 016285-005220US), which is incorporated byreference. Note that methods described herein for determining a cutoffvalue may be implemented when the reaction is sequencing, e.g. asdescribed in this section.

The determination of the fractional concentration of fetal DNA inmaternal plasma can also be done separate to the sequencing run. Forexample, the Y chromosome DNA concentration could be pre-determinedusing real-time PCR, microfluidics PCR or mass spectrometry. In fact,fetal DNA concentration could be determined using loci other than the Ychromosome and applicable to female fetuses. For example, Chan et alshowed that fetal-derived methylated RASSF1A sequences would be detectedin the plasma of pregnant women in the background of maternally derivedunmethylated RASSF1A sequences (Chan et al, Clin Chem 2006; 52:2211-8).The fractional fetal DNA concentration can thus be determined bydividing the amount of methylated RASSF1A sequences by the amount oftotal RASSF1A (methylated and unmethylated) sequences.

It is expected that maternal plasma would be preferred over maternalserum for practicing our invention because DNA is released from thematernal blood cells during blood clotting. Thus, if serum is used, itis expected that the fractional concentration of fetal DNA will be lowerin maternal plasma than maternal serum. In other words, if maternalserum is used, it is expected that more sequences would need to begenerated for fetal chromosomal aneuploidy to be diagnosed, whencompared with a plasma sample obtained from the same pregnant woman atthe same time.

Yet another alternative way of determining the fractional concentrationof fetal DNA would be through the quantification of polymorphicdifferences between the pregnant women and the fetus (Dhallan R, et al.2007 Lancet, 369, 474-481). An example of this method would be to targetpolymorphic sites at which the pregnant woman is homozygous and thefetus is heterozygous. The amount of fetal-specific allele can becompared with the amount of the common allele to determine thefractional concentration of fetal DNA.

In contrast to the existing techniques for detecting chromosomalaberrations, including comparative genomic hybridization, microarraycomparative genomic hybridization, quantitative real-time polymerasechain reaction, which detect and quantify one or more specificsequence(s), massively parallel sequencing is not dependent on thedetection or analysis of predetermined or a predefined set of DNAsequences. A random representative fraction of DNA molecules from thespecimen pool is sequenced. The number of different sequence tagsaligned to various chromosomal regions is compared between specimenscontaining or not containing tumoral DNA. Chromosomal aberrations wouldbe revealed by differences in the number (or percentage) of sequencesaligned to any given chromosomal region in the specimens.

In another example the sequencing technique on plasma cell-free DNA maybe used to detect the chromosomal aberrations in the plasma DNA for thedetection of a specific cancer. Different cancers have a set of typicalchromosomal aberrations. Changes (amplifications and deletions) inmultiple chromosomal regions may be used. Thus, there would be anincreased proportion of sequences aligned to the amplified regions and adecreased proportion of sequences aligned to decreased regions. Thepercentage representation per chromosome could be compared with the sizefor each corresponding chromosome in a reference genome expressed aspercentage of genomic representation of any given chromosome in relationto the whole genome. Direct comparisons or comparisons to a referencechromosome may also be used.

VIII. Mutation Detection

Fetal DNA in maternal plasma exists as a minor population, with anaverage of 3% to 6% of maternal plasma DNA being contributed by thefetus. Because of this reason, most of the previous work in the fieldhas focused on the detection of DNA targets which the fetus hasinherited from the father, and which are distinguishable from themajority maternal DNA background in maternal plasma. Examples of suchpreviously detected targets include the SRY gene on the Y chromosome (LoY M D et al. 1998 Am J Hum Genet, 62, 768-775) and the RHD gene when themother is RhD-negative (Lo Y M D et al. 1998 N Engl J Med, 339,1734-1738.

For fetal mutation detection, previous strategies using maternal plasmaare limited to autosomal dominant conditions in which the father is acarrier, the exclusion of autosomal recessive diseases by directmutation detection when the father and mother carries differentmutations, or by linkage analysis (Ding C. et al 2004 Proc Natl Acad SciUSA 101, 10762-10767). These previous strategies have significantlimitations. For example, for a couple where both the male and femalepartners carry the same mutation, then it would be impossible to carryout meaningful prenatal diagnosis by direct mutation detection inmaternal plasma.

Such a scenario is illustrated in FIG. 23. In this scenario, there willbe three possible fetal genotypes, NN, NM and MM, where N represents thenormal allele and M represents the mutant allele. Examples of mutantalleles include those causing cystic fibrosis, beta-thalassemia,alpha-thalassemia, sickle cell anemia, spinal muscular atrophy,congenital adrenal hyperplasia, etc. Other examples of such disorderscan be found in the Online Mendelian Inheritance in Man (OMIM)www.ncbi.nlm.nih.gov/sites/entrez?db=OMIM&itool=toolbar. In maternalplasma, most of the DNA will be coming from the mother and would be NM.For any of the three fetal genotypes, there will not be any unique fetalallele which would allow its unique detection in maternal plasma. Thus,the conventional strategy cannot be applied here.

Embodiments described herein allow handing such scenarios. In thescenario where the mother and fetus are both NM, then the N allele and Mallele will be in allelic balance. However, if the mother is NM and thefetus is NN, then there will be allelic imbalance in maternal plasma,with the N allele being overrepresented. On the other hand, if themother is NM and the fetus is MM, then there will be allelic imbalancein maternal plasma, with the M allele being overrepresented. Thus, forfetal mutation detection, the null hypothesis refers to the absence ofallelic imbalance when the fetus is of the NM genotype. The alternativehypothesis refers to the presence of allelic imbalance and the fetalgenotype could be NN or MM depending on whether the N or M allele isoverrepresented.

The presence or absence of allelic imbalance can be determined bydigital PCR using embodiments described herein. In a first scenario, aparticular volume of maternal plasma contains the DNA released from 100cells, in which 50 are from the mother and 50 are from the fetus. Thus,the fractional concentration of fetal DNA in this volume of plasma is50%. When the mother is of the genotype NM, then there will be 50 Nalleles and 50 M alleles contributed by the mother. If the fetus is ofthe genotype NM, then there will be 50 N alleles and 50 M allelescontributed by the fetus. Therefore, there will be no allelic imbalancebetween the N allele and the M allele, a total of 100 copies each. Onthe other hand, if the fetus is of the NN genotype, then there will be100 fetal-derived N alleles in this volume of plasma. Thus, there willbe a total of 150 N alleles to 50 M alleles. In other words, there willbe allelic imbalance between N and M, with N being overrepresented at aratio of 3:1 in relation to M.

In the converse situation, if the fetus is of the MM genotype, thenthere will be 100 fetal-derived M alleles in this volume of plasma.Thus, there will be 150 M alleles to 50 N alleles. In other words, therewill be allelic imbalance between N and M, with M being overrepresentedat a ratio of 3:1 in relation to N. Such allelic imbalance can bemeasured by digital PCR. The allele with the smaller number of positivewells is considered as the reference template. Similar to digitalRNA-SNP and digital RCD analyses, the actual distribution of the allelesin the digital PCR experiment would be governed by the Poissonprobability density function. Therefore, while the theoretical degree ofallelic imbalance in the present scenario is 3:1, the expected degree ofallelic imbalance would be dependent on the average templateconcentration per well during the digital PCR analysis. Thusinterpretation cutoffs, such as for SPRT analysis, appropriate for theaverage reference template concentration per well (m_(r)) would need tobe used for case classification.

Furthermore, the degree of allelic imbalance that needs to be measuredis dependent on the fractional fetal DNA concentration. In contrast tothe above example, let's consider a particular volume of maternal plasmacontains the DNA released from 100 cells, in which 90 are from themother and 10 are from the fetus. Thus, the fractional concentration offetal DNA in this volume of plasma is 10%. When the mother is of thegenotype NM, then there will be 90 N alleles and 90 M allelescontributed by the mother. If the fetus is of the genotype NM, thenthere will be 10 N alleles and 10 M alleles contributed by the fetus.Therefore, there will be no allelic imbalance between the N allele andthe M allele, a total of 100 copies each. On the other hand, if thefetus is of the NN genotype, then there will be 20 fetal-derived Nalleles in this volume of plasma. Thus, there will be a total of 110 Nalleles to 90 M alleles.

In other words, there will be allelic imbalance between N and M, with Nbeing overrepresented. In the converse situation, if the fetus is of theMM genotype, then there will be 20 fetal-derived M alleles in thisvolume of plasma. Thus, there will be 110 M alleles to 90 N alleles. Inother words, there will be allelic imbalance between N and M, with Mbeing overrepresented. The theoretical degree of allelic imbalance whenthe fetal DNA fractional concentration is 10% would be 110:90, which isdifferent to the 3:1 ratio when there is 50% fetal DNA as shown in theabove example. Thus interpretation cutoffs, such as for SPRT analysis,appropriate for the fetal DNA fractional concentration would need to beused for case classification.

Thus, plasma DNA will be extracted. The amount of maternal and fetal DNAin the plasma sample will be quantified, for example by the real-timePCR assays previously established (Lo, et al. 1998 Am J Hum Genet 62,768-775) or other types of quantifier well-known to those of skill inthe art, e.g. SNP markers (Dhallan R et al. 2007 Lancet, 369, 474-481)and fetal epigenetic markers (Chan K C A et al. 2006 Clin Chem, 52,2211-2218). The fetal DNA percentage will be calculated. Then thequantified plasma DNA sample is prepared (e.g. diluted or concentrated)such that during digital PCR analysis, each reaction well will containan average of one template molecule (can be either the N or M allele).The digital PCR analysis will be carried out using a pair of primers,plus two TaqMan probes, one specific to the N allele, while the otherone specific to the M allele. The number of wells which are positiveonly for M and the number of wells which are positive only for N will becounted. The ratio of these wells will be used to determine if there isevidence of allelic imbalance. Statistical evidence of allelic imbalancecan be sought by methods well-known to those of skill in the art, suchas using SPRT. In one variant of this analysis, it is also possible tocount the number of wells which are positive either for M only or for Mand N; as well as to count the number of wells which are positive eitherfor N only or for M and N; and to derive a ratio of these counts. Onceagain, statistical evidence of allelic imbalance can be sought bymethods well-known to those of skill in the art, such as using SPRT.

The dosage determination of fetal gene mutation, called digital relativemutant dosage (RMD), was validated using female/male (XX/XY) DNAmixtures. Blood cell DNA from a male and a female was each mixed withmale DNA, producing samples with XX or XY genotypes in a background ofXY at fractional concentrations of 25% and 50%, respectively, as shownin FIG. 24A.

In addition, blood cell samples were also obtained from 12 male and 12female subjects. The female blood cell DNA (genotype XX) was each mixedwith a 3-fold excess of male blood cell DNA (genotype XY), thusproducing 12 DNA mixtures with 25% of DNA with XX genotype in abackground of 75% DNA with XY genotype, with results shown in FIG. 24B.

An aim of the SPRT analysis was to determine the minor genotype presentin the background DNA. In DNA mixtures with 25% of XX DNA in abackground of 75% XY DNA, the minor allele would be the Y derived fromthe 75% of DNA. Since 25% of the DNA in the sample was of XX genotype,if there were a total of 200 molecules of DNA in the sample, then 150molecules would have originated from the XY individual. Hence, thenumber of Y alleles would be expected to be 75. The number of X allelescontributed by the male proportion of DNA (genotype XY) is also 75. Thenumber of X alleles contributed by the female (genotype XX) is 50 (2times 25). Therefore, X to Y ratio is 125/75=(1+25%)/(1−25%)=5/3.

For the second part of this study, blood cell samples were obtained frommale and female subjects carrying HbE (G→A) and CD41/42 (CTTT/−)mutations on the beta-globin gene, i.e., the hemoglobin, beta (HBB)gene. To mimic maternal plasma samples obtained from heterozygousmothers (MN, where M=mutant and N=wildtype) bearing male fetuses withall possible genotypes (MM, MN or NN), blood cell DNA from males whowere either homozygous for the wildtype alleles (NN) or heterozygous(MN) for one of the two mutations was each mixed with a blood cell DNAsample collected from females heterozygous for the same mutation (MN).DNA mixtures at various fractional male/mutant DNA concentrations werethus produced. Blood cell DNA sample from a female homozygous for theCD41/42 deletion (MM) was also used for preparing the DNA mixtures. Toensure an accurate male proportion for the SPRT classification, thefractional male DNA concentration of each DNA mixture was determinedusing the ZFY/X assay.

The digital ZFY/X assay was used for validating the SPRT as well asdetermining the fractional male DNA concentration in the DNA mixtures.The dosage of Zinc Finger protein sequences on chromosome X (ZFX) and Y(ZFY) was determined by digital PCR analysis. An 87-bp amplicon of theZFX and ZFY loci was first co-amplified by the forward primer5′-CAAGTGCTGGACTCAGATGTAACTG-3′ (SEQ ID NO:47) and the reverse primer5′-TGAAGTAATGTCAGAAGCTAAAACATCA-3′ (SEQ ID NO:48). Twochromosome-specific TaqMan probes were designed to distinguish betweenthe chromosome X and Y paralogs, and their sequences were5′-(VIC)TCTTTAGCACATTGCA(MGBNFQ)-3′ (SEQ ID NO:49) and5′-(FAM)TCTTTACCACACTGCAC(MGBNFQ)-3′ (SEQ ID NO:50), respectively.

The mutant dosage in the DNA mixtures was determined by digital PCRanalysis of the normal allele relative to the mutant allele. For the HbEmutation, an 87-bp amplicon of the normal and mutant alleles was firstco-amplified by the forward primer 5′-GGGCAAGGTGAACGTGGAT-3′ (SEQ IDNO:51) and the reverse primer 5′-CTATTGGTCTCCTTAAACCTGTCTTGTAA-3′ (SEQID NO:52). Two allele-specific TaqMan probes were designed todistinguish between the normal (G) and mutant (A) alleles, and theirsequences were 5′-(VIC)TTGGTGGTGAGGCC (MGBNFQ)-3′ (SEQ ID NO:53) and5′-(FAM)TGGTGGTAAGGCC (MGBNFQ)-3′ (SEQ ID NO:54), respectively. Resultsfor the HbE mutation are shown in FIG. 25.

For the CD41/42 deletion mutation, an 87- and 83-bp amplicon of thenormal and mutant alleles was first co-amplified by the forward primer5′-TTTTCCCACCCTTAGGCTGC-3′ (SEQ ID NO:55) and the reverse primer5′-ACAGCATCAGGAGTGGACAGATC-3′ (SEQ ID NO:56), respectively. Twoallele-specific TaqMan probes were designed to distinguish between thenormal (without deletion) and mutant (with deletion) alleles, and theirsequences were 5′-(VIC)CAGAGGTTCTTTGAGTCCT(MGBNFQ)-3′ (SEQ ID NO:57) and5′-(FAM)AGAGGTTGAGTCCTT(MGBNFQ)-3′ (SEQ ID NO:58), respectively. Resultsfor the HbE mutation are shown in FIGS. 26A and 26B.

These experiments were carried out on the BioMark™ System (Fluidigm)using the 12.765 Digital Arrays (Fluidigm). The reaction for one panelwas set up using 2×TaqMan Universal PCR Master Mix (Applied Biosystems)in a reaction volume of 10 μL. For the CD41/42 and ZFY/X assays, eachreaction contained 1×TaqMan Universal PCR Master Mix, 900 nM of eachprimer, 125 nM of each probe and 3.5 μL of DNA mixture at 1 ng/μL. Forthe HbE assay, 250 nM and 125 nM of probes targeting the normal (G) andmutant (A) alleles were added, respectively. The sample/assay mixturewas loaded into the Digital Array by the NanoFlex™ IFC controller(Fluidigm). The reaction was carried out on the BioMark™ System forsignal detection. The reaction was initiated at 50° C. for 2 min,followed by 95° C. for 10 min and 50 cycles of 95° C. for 15 s and 57°C. (for ZFY/X and CD41/42) or 56° C. (for HbE) for 1 min. At least onereaction panel was used for each case, and data were aggregated fromextra panels for samples which remained unclassified until a decisioncould be made.

It will also be obvious to those of skill in the art that the digitalPCR can be performed using methods well-known to those of skill in theart, e.g. microfluidics chips, nanoliter PCR microplate systems,emulsion PCR, polony PCR, rolling-circle amplification, primer extensionand mass spectrometry.

IX. Example with Cancer

In one embodiment, the present invention may be performed to classify asample as having allelic ratio skewing or not, as may occur in acancerous tumor. In one aspect, for each case, the number of wells withpositive signal for the A allele only, the G allele only, and bothalleles were determined by digital PCR. The reference allele was definedas the allele with the smaller number of positive wells. (In theunlikely scenario that both alleles have the same number of positivewells, then either can be used as the reference allele.) The inferredaverage concentration of the reference allele per well (m_(r)) wascalculated using the total number of wells negative for the referenceallele, irrespective whether the other allele was positive, according tothe Poisson probability density function. We use a hypothetical exampleto illustrate the calculation.

In a 96-well reaction, 20 wells are positive for the A allele, 24 wellsare positive for the G allele, and 28 wells are positive for bothalleles. The A allele would be regarded as the reference allele becauseless wells are positive for this allele. The number of wells negativefor the reference allele would be 96−20−28=48. Therefore, m_(r) can becalculated using the Poisson distribution and would be −ln(48/96)=0.693.

In the context of LOH detection, the null hypothesis refers to a samplethat is assumed to lack allelic ratio skewing caused by the presence ofa deletion of one allele. Under this assumption, the expected ratio ofthe number of positive wells for the two alleles would be 1:1 and, thus,the expected proportion of informative wells (wells positive for onlyone allele) containing the potentially overrepresented allele would be0.5.

In the context of LOH detection, the alternative hypothesis refers to asample that is assumed to have allelic ratio skewing caused by thepresence of a deletion of one allele in 50% of the cells of the sample.As the allelic ratio between the overrepresented allele and thereference allele is 2:1, the average concentration of theoverrepresented allele per well would be doubled that of the referenceallele. However, the number of wells positive for the overrepresentedallele would not be simply two times that for the reference allele butwould follow the Poisson distribution.

An informative well is defined as a well positive for either the A orthe G allele but not for both alleles. The calculation of the expectedproportion of the number of wells containing the overrepresented allelesfor samples with allelic ratio skewing is the same as is shown in Table600. In the above example, if LOH is present in 50% of tumor cells, theaverage concentration of the G allele per well would be 2 times0.693=1.386. If LOH is present in more than 50% of the tumor cells, thenthe average concentration of the G allele per well would be according tothe formula: 1/[1−(proportion with LOH)]×m_(r).

The expected proportion of wells positive for the G allele would be1−e^(−1 . . . 386)=0.75 (i.e., 75% or 72 wells). Assuming that thepositivity of a well for the A or G allele is independent,0.5×0.75=0.375 of the wells would be positive for both the A and Galleles. Hence, 0.5−0.375=0.125 of the wells would be positive for the Aallele only and 0.75−0.375=0.375 of the wells would be positive for theG allele only. Therefore, the proportion of informative wells would be0.125+0.375=0.5. The expected proportion of informative wells carryingthe G allele would be 0.375/0.5=0.75. This expected value for P_(r) canthen be used for the construction of appropriate SPRT curves fordetermining whether allelic ratio skewing (i.e. LOH in this context) ispresent in the sample.

The actual proportion of informative wells carrying the non-referenceallele experimentally determined by the digital PCR analysis (P_(r)) wasthen used to determine whether the null or alternative hypothesis wouldbe accepted, or whether further analysis with more wells would benecessary. The decision boundaries for P_(r) to accept the null oralternative hypothesis was calculated based on a threshold likelihoodratio of 8 as this value had been shown to provide satisfactoryperformance to discriminate samples with and without allelic imbalancein the context of cancer detection (Zhou, W, et al. (2001) NatBiotechnol 19, 78-81; Zhou et al 2002, supra). In the above example, thenumber of informative wells would be 20+24=44 and the experimentallyobtained P_(r) would be 24/44=0.5455. The decision boundaries would be≤0.5879 to accept the null hypothesis and ≥0.6739 to accept thealternative hypothesis. Therefore, the sample in this example would beclassified as NOT having allelic ratio skewing.

In conclusion, we outlined an approach to detect sequence imbalance in asample. In one embodiment, this invention can be used for thenoninvasive detection of fetal chromosomal aneuploidy, such as trisomy21 by analysis of fetal nucleic acids in maternal plasma. This approachcan also be applied to other biological materials containing fetalnucleic acids, including amniotic fluid, chorionic villus samples,maternal urine, endocervical samples, maternal saliva, etc. First, wedemonstrated the use of this invention for determining allelic imbalanceof a SNP on PLAC4 mRNA, a placenta-expressed transcript on chromosome21, in maternal plasma of women bearing trisomy 21 fetuses. Second, wedemonstrated that our invention can be used as a non-polymorphism basedmethod, through relative chromosome dosage (RCD) analysis, for thenoninvasive prenatal detection of trisomy 21. Such a digital RCD-basedapproach involves the direct assessment of whether the total copy numberof chromosome 21 in a sample containing fetal DNA is overrepresentedwith respect to a reference chromosome. Even without elaborateinstrumentation, digital RCD allows the detection of trisomy 21 insamples containing 25% fetal DNA. We applied the sequential probabilityratio test (SPRT) to interpret the digital PCR data. Computer simulationanalyses confirmed the high accuracy of the disease classificationalgorithm.

We further outlined that the approach can be applied to thedetermination of other forms of nucleic acid sequence imbalances otherthan chromosomal aneuploidy, such as that for the detection of fetalmutation or polymorphism detection in maternal plasma and regional gainsand losses in the genomes of malignant cells through the analysis oftumor-derived nucleic acids in plasma.

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a computer readable medium for storage and/ortransmission, suitable media include random access memory (RAM), a readonly memory (ROM), a magnetic medium such as a hard-drive or a floppydisk, or an optical medium such as a compact disk (CD) or DVD (digitalversatile disk), flash memory, and the like. The computer readablemedium may be any combination of such storage or transmission devices.

Such programs may also be encoded and transmitted using carrier signalsadapted for transmission via wired, optical, and/or wireless networksconforming to a variety of protocols, including the Internet. As such, acomputer readable medium according to an embodiment of the presentinvention may be created using a data signal encoded with such programs.Computer readable media encoded with the program code may be packagedwith a compatible device or provided separately from other devices(e.g., via Internet download). Any such computer readable medium mayreside on or within a single computer program product (e.g. a hard driveor an entire computer system), and may be present on or within differentcomputer program products within a system or network. A computer systemmay include a monitor, printer, or other suitable display for providingany of the results mentioned herein to a user.

An example of a computer system is shown in FIG. 27. The subsystemsshown in FIG. 27 are interconnected via a system bus 2775. Additionalsubsystems such as a printer 2774, keyboard 2778, fixed disk 2779,monitor 2776, which is coupled to display adapter 2782, and others areshown. Peripherals and input/output (I/O) devices, which couple to I/Ocontroller 2771, can be connected to the computer system by any numberof means known in the art, such as serial port 2777. For example, serialport 2777 or external interface 2781 can be used to connect the computerapparatus to a wide area network such as the Internet, a mouse inputdevice, or a scanner. The interconnection via system bus allows thecentral processor 2773 to communicate with each subsystem and to controlthe execution of instructions from system memory 2772 or the fixed disk2779, as well as the exchange of information between subsystems. Thesystem memory 2772 and/or the fixed disk 2779 may embody a computerreadable medium.

The above description of exemplary embodiments of the invention has beenpresented for the purposes of illustration and description. It is notintended to be exhaustive or to limit the invention to the precise formdescribed, and many modifications and variations are possible in lightof the teaching above. The embodiments were chosen and described inorder to best explain the principles of the invention and its practicalapplications to thereby enable others skilled in the art to best utilizethe invention in various embodiments and with various modifications asare suited to the particular use contemplated.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method for determining whether a nucleic acidsequence imbalance exists within a biological sample of a female subjectpregnant with a fetus having a fetal genome, wherein the biologicalsample including a mixture of cell-free nucleic acid molecules from thefemale subject and from the fetus, the method comprising: separating thecell-free nucleic acid molecules of the biological sample into aplurality of reactions; measuring signals from the plurality ofreactions; receiving, at a computer system, quantitative data includingthe measured signals from the plurality of reactions involving thecell-free nucleic acid molecules from the biological sample, wherein thedata includes: for a plurality of clinically relevant nucleic acidsequences: (1) first quantitative data indicating a first total amountof the plurality of clinically relevant nucleic acid sequences in theplurality of reactions; and for a plurality of background nucleic acidsequences: (2) second quantitative data indicating a second total amountof the plurality of background nucleic acid sequences in the pluralityof reactions, wherein the plurality of background nucleic acid sequencesare different from any one of the clinically relevant nucleic acidsequences; determining, by the computer system, the first total amountof the plurality of clinically relevant nucleic acid sequences from thefirst quantitative data; determining, by the computer system, the secondtotal amount of the plurality of background nucleic acid sequences fromthe second quantitative data; determining a parameter from the firsttotal amount and the second total amount, wherein the parameter providesa relative amount between the first total amount and the second totalamount; comparing the parameter to one or more cutoff values; and basedon the comparison, determining a classification of whether the nucleicacid sequence imbalance exists in the fetal genome for the plurality ofclinically relevant nucleic acid sequences.
 2. The method of claim 1,wherein determining the first total amount from the first quantitativedata includes: for each of the clinically relevant nucleic acidsequences: analyzing the first quantitative data to determine arespective first amount of the clinically relevant nucleic acid sequencein the plurality of reactions, wherein determining the second totalamount from the second quantitative data includes: for each of thebackground nucleic acid sequences: analyzing the second quantitativedata to determine a respective second amount of the background nucleicacid sequence in the plurality of reactions, calculating the first totalamount by summing the respective first amounts; and calculating thesecond total amount by summing the respective second amounts.
 3. Themethod of claim 2, further comprising: correcting the respective firstamounts and the respective second amounts before calculating the firsttotal amount and the second total amount.
 4. The method of claim 1,wherein the plurality of reactions are sequencing reactions.
 5. Themethod of claim 4, wherein the sequencing reactions involve ligation. 6.The method of claim 4, wherein the sequencing reactions involvesynthesis.
 7. The method of claim 4, wherein sequence tags are obtainedfrom the sequencing reactions, and wherein determining the first totalamount includes: aligning the sequence tags to a human genome; andcounting a number of sequence tags that align to the plurality ofclinically relevant nucleic acid sequences.
 8. The method of claim 4,further comprising: enriching the biological sample for the plurality ofclinically relevant nucleic acid sequences and the plurality ofbackground nucleic acid sequences; and subsequently, performing asequencing of nucleic acid molecules in the enriched biological sample.9. The method of claim 8, wherein enriching the biological sample forthe plurality of clinically relevant nucleic acid sequences and theplurality of background nucleic acid sequences includes: using ahybridization-based technique.
 10. The method of claim 9, wherein usingthe hybridization-based technique includes: using an oligonucleotidearray to select the plurality of clinically relevant nucleic acidsequences and the plurality of background nucleic acid sequences viahybridization.
 11. The method of claim 8, wherein enriching thebiological sample for the plurality of clinically relevant nucleic acidsequences and the plurality of background nucleic acid sequencesincludes: amplifying the plurality of clinically relevant nucleic acidsequences and the plurality of background nucleic acid sequences. 12.The method of claim 11, wherein a same primer pair is used to amplifyone of the clinically relevant nucleic acid sequences and one of thebackground nucleic acid sequences.
 13. The method of claim 1, furthercomprising: prior to performing the plurality of reactions, enrichingthe biological sample for the plurality of clinically relevant nucleicacid sequences and the plurality of background nucleic acid sequences.14. The method of claim 1, further comprising: determining a fractionalconcentration of fetal DNA in the biological sample; and using thefractional concentration of fetal DNA to determine the one or morecutoff values.
 15. The method of claim 14, wherein determining thefractional concentration of fetal DNA in the biological sample includesa quantification of a polymorphic difference between the female subjectand the fetus.
 16. The method of claim 15, wherein the quantificationincludes: identifying a target polymorphic site at which the femalesubject is homozygous and the fetus is heterozygous; and comparing anamount of a fetal-specific allele at the target polymorphic site and anamount of a common allele at the target polymorphic site to determinethe fractional concentration of fetal DNA, the common allele not beingfetal-specific.
 17. The method of claim 14, wherein the fetus is male,and wherein determining the fractional concentration of fetal DNA in thebiological sample includes: determining a Y chromosome DNAconcentration.
 18. The method of claim 14, wherein determining thefractional concentration of fetal DNA in the biological sample includes:comparing an amount of DNA molecules exhibiting a fetal-specificmethylation pattern at a first locus to a total amount of DNA moleculesat the first locus.
 19. The method of claim 18, wherein fetal-derivedDNA molecules are hypermethylated and maternally-derived DNA moleculesare hypomethylated at the first locus.
 20. The method of claim 1,wherein each reaction indicates a presence or absence of a clinicallyrelevant nucleic acid sequence of the plurality of clinically relevantnucleic acid sequences and a background nucleic acid sequence of theplurality of background nucleic acid sequences, wherein the firstquantitative data provides a number of the plurality of reactionspositive for the plurality of clinically relevant nucleic acidsequences, and wherein the second quantitative data provides a number ofthe plurality of reactions positive for the plurality of backgroundnucleic acid sequences.
 21. The method of claim 1, wherein theclinically relevant nucleic acid sequences are of a first chromosome,wherein the background nucleic acid sequences are of one or morereference chromosomes that are different than the first chromosome, andwherein the classification is of whether the nucleic acid sequenceimbalance exists in the fetal genome for the first chromosome.
 22. Themethod of claim 1, wherein the parameter includes a ratio between thefirst total amount and the second total amount.
 23. The method of claim1, wherein the biological sample is plasma.
 24. The method of claim 1,wherein the plurality of clinically relevant nucleic acid sequences arenon-consecutive and non-polymorphic.
 25. The method of claim 24, whereinthe plurality of background nucleic acid sequences are non-polymorphic.